Nucleic acid and polypeptide linked to breast cancer and uses therefor

ABSTRACT

The present invention relates to an isolated domain of G3BP-2 that mediates binding between G3BP-2 and other proteins, and nucleic acids encoding same. The invention also relates to a method for diagnosing, treating and preventing breast cancer including the step of using a nucleic acid and/or encoded polypeptide for G3BP-2, or fragment thereof, to detect, treat or prevent breast cancer in a mammal, preferably human. In one particular form, the invention relates to an antigen presenting cell, preferably a dendritic cell, that is capable of presenting G3BP-2 or fragments thereof. The invention also relates to lymphocytes, in particular cytotoxic T-lymphocytes, that are G3BP-2 antigen specific.

FIELD OF THE INVENTION

THIS INVENTION relates to an isolated domain of G3BP-2 that mediatesbinding between G3BP-2 and other proteins, and nucleic acids encodingsame. More particularly, a method for diagnosing, treating andpreventing breast cancer including the step of using a nucleic acidand/or encoded polypeptide for G3BP-2 to detect, treat or prevent breastcancer in a mammal.

BACKGROUND OF THE INVENTION

ras-GTPase-Activating Protein SH3-Domain-binding Proteins (G3BPs) are afamily of proteins which comprise SH3 domain-binding motifs which havebeen shown to specifically bind the ras-GTPase-activating protein,rasGAP¹²⁰ (Parker et al., 1996; Kennedy et al, 1997). Furthermore, thisfamily of proteins have been shown to be RNA-binding proteins (Kennedyet al., 1997) that may have an RNAase activity on c-myc transcripts(Gallouzi et al., 1998). Both the ras-GAP signalling pathway and c-mychave been implicated in oncogenic activity (Bos, 1989; Facchini andPenn, 1998). The evidence presented suggests that the G3BP family ofproteins are members of a novel signal transduction mechanism thatutilises components of previously described pathways to regulate mRNAstability and through these pathways may regulate oncogenic signals orfactors. These activities may be modulated through their SH3domain-binding activity (Parker et al., 1996), their RNAase activity(Gallouzi et al., 1998) or their helicase activity (Costa et al., 1999).G3BPs have recently been shown to be upregulated at the transcriptionallevel in some cancers (Guitard et al., 2001).

rasGAP¹²⁰ is an important regulator of signal transduction (Pomerance etal., 1996) as it sits at the nexus of positive and negative control ofthe oncogene ras. rasGAP¹²⁰ itself stimulates the hydrolysis of GTPbound ras (reviewed in Tocque et al., 1997) and thereby regulates theactivity of ras. The amino-terminus of rasGAP¹²⁰ comprises a Scrhomology (SH3) domain (Tocque et al. 1997) which has been implicated inan effector-like activity (Duchesne et al., 1993).

Human G3BP-1 was first identified by its co-immunoprecipitation withrasGAP¹²⁰ using an antibody raised to the carboxy-terminal domain ofrasGAP¹²⁰ (Parker et al., 1996). G3BP was the first protein shown tobind the rasGAP¹²⁰ SH3 domain, however, other rasGAP¹²⁰ SH3 bindingproteins have since been reported, including a 14 kDa protein (Hu andSettleman 1997) and the huntingtin protein (Liu et al., 1997). Geneticstudies in Drosophila support a role for G3BP in ras signaling (Pazmanet al., 2000).

The inventors previously cloned and sequenced mouse G3BP-2 as part of ageneral screening for RNA Recognition motif (RRM)-containing proteins(Kennedy et al., 1997). Primary sequence analysis of G3BPs alsoindicated that they contain an RNA Recognition Motif (RRM) (Nagai etal., 1995), an RGG domain (Burd and Dreyfuss 1994; Siomi and Dreyfuss1997) and a Nuclear Transport Factor 2-like (NTF2-like) domain (Suyamaet al., 2000). The proposed structure of the RRM in G3BP has beenreported elsewhere (Kennedy et al. 1997). The G3BPs also containacid-rich and RGG domains which are often considered auxiliary domainsfor RRM-type RNA-binding proteins (Burd and Dreyfuss 1994; Siomi andDreyfuss 1997). These structural motifs are consistent with a recentfinding that G3BP-1 is implicated in RNA metabolism by acting in vitroas a cleavage factor for c-myc transcripts (Gallouzi et al. 1998).

NTF2 polypeptide is involved in nuclear transport of polypeptides andappears to be facilitated by binding RanGDP in the cytoplasm. OnceNTF2/RanGDP is bound to a cargo the complex is imported to the nucleuswhere it is released and the Ran nucleotide exchange factor, RCC1,converts RanGDP to RanGTP. This signals export of NTF2 to the cytoplasmwhere RanGTP is hydrolysed by Ran GTPase activating protein (RanGAP) andthe system is reset (reviewed in Macara 1999). The NTF2-like domain ofG3BP-2 may target G3BP-2 to the nuclear envelope, although a mechanismfor this activity is unclear (Prigent et al., 2000).

RNA processing is an integral part of cellular metabolism controlledthrough pre-mRNA splicing, RNA transport and RNA stability (Dreyfuss etal., 1996). Regulation of RNA metabolism has been shown to play animportant role in development. Recently there has been increasedinterest in the control of mRNA translation mediated by RNA-bindingproteins, in particular the role of these proteins in 5′ UTRinteractions that influence elongation factors (Svitkin et al., 1996) aswell as 3′ interactions involving translational activity (Dreyfuss etal. 1996) and degradation (Gallouzi et al., 1998). It is important tocharacterise the mechanisms that allow RNA-binding proteins to respondto environmental and developmental signals through transduction cascadesin order to understand their role in human diseases.

SH3 domains were initially characterised in signal transduction proteinssuch as Src, Fyn and Grb as well as rasGAP¹²⁰. Typically these domainsinteract with proline rich motifs with a minimum consensus of PxxP(Urquhart et al., 2000 and papers cited therin). It has also been shownthat the acidic and PxxP domains, and not the RNA-binding domain nor theNTF2-like domain of G3BP-2, are sufficient to mediate binding to IκBα(Prigent et al; 2000). Primary sequence analysis of alternativelyspliced homologues of human G3BP-2a and G3BP-2b reveals that theyrespectively comprise five and six minimal potential SH3 domain-bindingmotifs (Lee et al., 1996). As G3BP-2a and G3BP-2b comprise PxxPsequences, it was predicted that these proline-rich motifs would bindwith SH3 domains of polypeptides.

Major advances have been achieved in the early diagnosis (screeningmammography) and treatment (adjuvant therapy) of breast cancer and thishas translated into a significant reduction in the mortality generatedby this disease (Chlebowski, 2002). However, breast cancer stillaccounts for 26% of the cancers diagnosed in Australia in 1999, and in1996 there were 9,556 new cases diagnosed with 2,619 deaths ((AIHW),1999). An additional important input into the better management of thedisease has been the characterization of genetic predisposing markersBRCA1 and 2 (reviewed in (Nathanson et al., 2001)), allowing theprediction of a proportion (10%) of women at risk to develop breastcancer. Considering the reduced adjuvant therapeutic options currentlyavailable (Pritchard et al., 2002) and the risks involved with theiruse, novel strategies are urgently needed that could prevent thedevelopment of the disease at early stages of the disease and/or inthose with higher genetic risk.

SUMMARY OF THE INVENTION

Although G3BPs have been shown to bind the SH3 domain of GAP¹²⁰ theinventors were surprised to discover that this binding is mediatedthrough the N-terminal NTF2-like domain of G3BP and not facilitated by aproline-rich motif (PxxP) contained within G3BP-2 as the prior art wouldsuggest. The smallest G3BP truncated protein that was capable of bindingto the SH3 domain of rasGAP¹²⁰ did not contain any of the predicted PxxPmotifs normally associated with SH3 binding. The unexpected resultsclearly showed that the N-terminal NTF2-like domain of G3BP isresponsible for the binding interactions with N-terminal rasGAP¹²⁰. Thisfinding has led to novel uses of G3BP, in particular the NTF2-likedomain thereof, as described herein for identifying and producingpotential reagents for diagnosing, treating or preventing breast cancer.

In a first aspect, the invention provides an isolated G3BP-2 proteinfragment comprising an NTF2-like domain, said isolated G3BP2 proteinfragment capable of binding another protein by way of said NTF2-likedomain.

Preferably, the G3BP-2 protein comprises G3BP-2a and/or G3BP-2bproteins.

Preferably, the another protein is selected from the group consistingof: ran nuclear pore polypeptide, ubiquitin hydrolase and GAP¹²⁰.

More preferably, the ubiquitin hydrolase is ODE1.

The NTF2-like domain preferably is encoded by amino acid residues 1 to146 as set forth in SEQ ID NO: 22.

In a second aspect the invention provides an isolated protein complexcomprising a G3BP-2 protein having an NTF2-like domain and anotherprotein bound to the NTF2-like domain, selected from the groupconsisting of: ran nuclear pore polypeptide, ubiquitin hydrolase andGAP¹²⁰.

In a third aspect, the invention provides an isolated G3BP-2 protein,inclusive of a fragment, homolog, variant or derivative thereof capableof eliciting an immune response in an animal.

Preferably, the animal is human.

Preferably, the G3BP-2 fragment is selected from the group consistingof: (i) KLPNFGFW; [SEQ ID NO:1] (ii) IMFRGEVRL; [SEQ ID NO:2] and (iii)SATPPPAEPASLPQEPPKPRV [SEQ ID NO:3]

In a fourth aspect, the invention provides an isolated G3BP-2 proteinfragment selected from the group consisting of: (a) KLPNFGFW; [SEQ IDNO:1] (b) IMFRGEVRL; [SEQ ID NO:2] and (c) SATPPPAEPASLPQEPPKPRV [SEQ IDNO:3]

In a fifth aspect, the invention provides an isolated nucleic acidencoding a protein of the first aspect, inclusive of fragments,homologs, variants and derivatives thereof, each capable of bindinganother protein by way of said NTF2-like domain.

In one form, the isolated nucleic acid encodes a protein comprising theNTF2-like domain comprising an amino acid sequence as set forth in SEQID NO: 22, said NTF2-like domain being encoded by amino acid residues 1to 146, wherein amino acid residue 1 is the first methionine (M).

In another form, the isolated nucleic acid comprises a nucleotidesequence set forth in SEQ ID NO: 23.

In a sixth aspect, the invention provides an isolated nucleic acidencoding a G3BP-2 protein fragment of the fourth aspect.

In a seventh aspect, the invention provides an expression vectorcomprising a nucleic acid of any one of the abovementioned aspects.

In an eighth aspect, the invention relates to use of an antagonist toprevent or disrupt binding between G3BP-2 and another protein.

In one form, the antagonist of the eighth aspect prevents or disruptsbinding between a NTF2-like domain of G3BP-2 and said another protein.

In another form, the antagonist is a mimetic of the NTF2-like domain ofG3BP-2.

In yet another form, the antagonist binds to the NTF2-like domain.

The antagonist may be a protein.

In one form, the protein comprises an Src homology 3 (SH3) domain.

Preferably, the protein comprises an amino acid sequence as set forth inSEQ ID NO: 6.

The antagonist may be a non-peptide compound.

In a ninth aspect, the invention provides an isolated antigen presentingcell which has been in contact with a G3BP-2 protein, fragment, homolog,variant or derivative thereof, wherein contact includes pulsing orloading the antigen presenting cell with G3BP-2 protein, fragment,homolog, variant or derivative thereof.

In a tenth aspect, the invention provides an isolated antigen presentingcell which has been transfected with a nucleic acid encoding G3BP-2protein, inclusive of fragments, homologs, variants and derivativesthereof.

The isolated antigen presenting cell of the ninth and tenth aspects ispreferably a dendritic cell.

The G3BP-2 protein, inclusive of a fragment, a homolog, a variant and aderivative thereof of the ninth and tenth aspects preferably comprisesan amino acid sequence as set forth in SEQ ID NO: 5.

The G3BP-2 fragment of the ninth and tenth aspects preferably comprisesan amino acid sequence selected from the group consisting of: KLPNFGFVV[SEQ ID NO: 1] and IMFRGEVRL [SEQ ID NO: 2].

In an eleventh aspect, the invention provides an isolated lymphocytecell that is G3BP-2 antigen specific.

Preferably, the isolated lymphocyte cell is a cytotoxic T-lymphocyte.

Preferably, the lymphocyte cell is G3BP-2 antigen specific for aprotein, inclusive of fragments, homologs, variants and derivativesthereof, comprising an amino acid sequence as set forth in SEQ ID NO: 5.

Preferably, the G3BP-2 protein fragment comprises an amino acid sequenceas set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

In a twelfth aspect, the invention provides a pharmaceutical compositioncomprising at least one active, wherein the active is selected from thegroup consisting of: a protein, a nucleic acid or an isolated cell ofany one of the above aspects.

In a thirteenth aspect, the invention provides a method for preventingor treating breast cancer in a mammal including the step ofadministering to said mammal a pharmaceutical composition comprising atleast one active, wherein the active is selected from the groupconsisting of: a protein, a nucleic acid, a mimetic of the NTF2-likedomain of G3BP-2, an antagonist that prevents or disrupts bindingbetween a NTF2-like domain of G3BP-2 and another protein or isolatedcell of any one of the above-mentioned aspects.

Preferably the mammal is human.

In a fourteenth aspect, the invention provides a method for modulatingcell proliferation including the step of administering to an animal orisolated cell, an active which prevents or disrupts binding betweenG3BP-2 and another protein.

Preferably the animal is human.

In a fifteenth aspect, the invention provides a method for isolating amolecule that binds G3BP-2, including the step of determining if one ormore candidates in a sample bind to the NTF2-like domain of G3BP-2.

In one form, the molecule is an antagonist.

The antagonist may be a protein or a non-protein molecule.

In a sixteenth aspect, the invention provides a method for diagnosingbreast cancer in a mammal including the steps of comparing G3BP-2protein expression in a test sample obtained from the mammal with G3BP-2in a reference sample, wherein if the expression of G3BP-2 in the testsample is different than the reference sample, the mammal is diagnosedwith an increased likelihood of having breast cancer.

G3BP-2 protein expression may be detected using an antibody.

The antibody may bind to a G3BP-2 protein, inclusive of a fragment, ahomolog, a variant and a derivative thereof, comprising an amino acidsequence as set forth in SEQ ID NO: 5.

In one form, the antibody binds to a G3BP-2 protein fragment comprisingan amino acid sequence SATPPPAEPASLPQEPPKPRV [SEQ ID NO: 3].

The G3BP-2 fragment may comprise a NTF2-like domain.

Preferably, the mammal is human.

In a seventeenth aspect, the invention provides a method for diagnosingbreast cancer in a mammal including the step of detecting a G3BP-2nucleic acid or fragment thereof in a test sample obtained from themammal.

Preferably, the test sample is breast tissue.

Preferably, the mammal is human.

In an eighteenth aspect, the invention provides a method of immunising amammal against breast cancer, including the step of administering tosaid mammal an immunogenic agent comprising at least one active selectedfrom the group consisting of:

-   -   (1) a G3BP-2 protein;    -   (2) a fragment, a homolog, a variant or a derivative of (1);    -   (3) a G3BP-2 nucleic acid;    -   (4) a fragment, a homolog, a variant or a derivative of (3);    -   (5) an isolated antigen presenting cell that has been contacted        with (1) or (2); and    -   (6) an isolated antigen presenting cell that has been        transfected with a nucleic acid of (3) or (4).

The immunisation may be preventative or as a treatment for an animalwith breast cancer.

The G3BP-2 protein fragment is preferably selected from the groupconsisting of: KLPNFGFVV [SEQ ID NO: 1] and IMFRGEVRL [SEQ ID NO: 2].

The antigen presenting cell is preferably a dendritic cell.

Preferably, the mammal is human.

The isolated protein and nucleic acid, and methods according to theaforementioned aspects of the invention are useful in therapeutic orprophylactic treatments of breast cancer and diagnosis thereof. As willbe described in more detail hereinafter, disruption of interactionsbetween G3BP-2 and another protein or endogenous binding partner mayinhibit tumour proliferation.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1 is a nucleotide sequence of HsaG3BP-2a [SEQ ID NO: 4] and encodedamino acid sequence [SEQ ID NO: 5]; and

FIG. 2 is an amino acid sequence [SEQ ID NO: 6] of the N-terminus ofrasGAP¹²⁰, which includes a SH3 domain;

FIG. 3 shows a clustal alignment of the G3BP family of proteins, mouseMmuG3BP2a [SEQ ID NO:7], human HsaG3BP2a [SEQ ID NO:5], mouse MmuG3BP2b[SEQ ID NO:8], human HsaG3PB2b [SEQ ID NO:9], human HsaG3BP1 [SEQ IDNO:10] and mouse MmuG3BP1 [SEQ ID NO:11];

FIG. 4 shows analysis of protein-protein interactions between the SH3domain of N-terminal rasGAP¹²⁰ and G3BP;

FIG. 5 shows Western blot analysis of G3BP expression in differenttissues;

FIGS. 6A and 6B shows immunohistochemistry of adult mouse tissues probedwith anti-G3BP-1 and anti-G3BP-2 antibodies;

FIGS. 7A and 7B show G3BP-2 immunohistochemistry of two human breastcancers. Panel A is an in situ ductal tumour and panel B shows aninfiltrating cancer;

FIG. 8A-T shows immunohistochemical staining of breast tumour sectionsand immunofluorescence of synchronised NIH 3T3 cells with antibodiesspecific for G3BPs. Panels A-C are breast tumour sections stained usingan antibody specific for G3BP-1. Panels A and B represent stained IDCwhile. Panel C shows a small section of stained normal ducts (ND).Panels D-O show immunohistochemistry of G3BP-2 in human breast tumours.Panel D shows a normal lobe, Panels E and F show normal ducts cuttransverse and longitudinally, respectively (CT denotes connectivetissue). Panels G and H show a normal duct adjacent to an IDC (DCdenotes ductal carcinoma). Panel I shows an IDC which does not expressG3BP-2. Panel J shows an IDC adjacent to normal connective tissue. Thearrow indicates cells within the connective tissue which stain positivefor G3BP-2 in the nucleus. Panel K shows a lower magnification of an IDC(left side) adjacent to normal connective tissue. Panels L-O illustratea variety of G3BP-2 subcellular localisations in human breast cancer.All panels are ductal carcinomas from different patients. Panel L showscytoplasmic localisation of G3BP-2. Panels M and N show nuclearlocalisation of G3BP-2 in two different cases of breast cancer;cytoplasmic expression is also observed in these sections. Panel O showsG3BP-2 expression around the nuclear envelope region; cytoplasmicstaining is also observed. Panels P-T show the immunofluorescence ofsynchronised NIH 3T3 cells. Panel P shows the sub-cellular localisationof G3BP-2 in cells in G0 phase (time=0). The time after serumstimulation and hence cell cycle commencement is 2 hours, 5 hours, 9hours and 12 hours for Panels Q, R, S and T, respectively.

FIG. 9 illustrates antibody specificity of G3BP-2 antibodies. The breastcancer cell line MDA-MB-435 and the cervical cancer cell line HeLa(labeled 435 and HeLa respectively) were lysed and equal amounts ofprotein were resolved by SDS-PAGE. The samples were transferred to amembrane and probed with either the polyclonal G3BP-2 antibody or thecommercial G3BP-1 antibody as indicated (Panel A). Purified recombinantG2BP-2b (lane 1), G3BP-1 (lane 2) and G3BP-2a (lane 3) were resolved bySDS-PAGE along with four different truncations of G3BP-2a, N1 (lane 4),C1 (lane 5), C2 (lane 6), N2 (lane 7). These samples were transferred toa membrane and probed with the G3BP-2 antibody (Panel B). Panel C is aschematic representation of G3BP-2a and the recombinant G3BP-2atruncations (N1, N2, C1, C2). It also illustrates the region in whichthe G3BP-2 polyclonal antibody binds. Inset shows the sub-domains withinG3BP-2a.;

FIG. 10 shows G3BP-2 peptide binding to HLA-A*0201 measured by T2binding assay; and

FIG. 11 is a graph showing data for a chromium release cytotoxic assayusing different effector cell: target cell ratios.

Table 1: shows exemplary conservative substitutions in the polypeptide;and

-   -   Table 2 summarises expression of G3BP-2 in 58 breast tumour        sections. DCIS=ductal carcinoma in situ, IDC=infiltrating ductal        carcinoma, LCIS=lobular carcinoma in situ, ILC=infiltrating        lobular carcinoma. Grade is assigned according to a range of        factors—a well-differentiated tumour is generally assigned a        grade 1 while a poorly differentiated tumour is assigned a        grade 3. ER=oestrogen receptor status. Node status refers to the        presence (+) or absence (−) of tumour in the lymph nodes. NG=not        graded. ND=not determined. The column labelled cytoplasm        indicates the level of expression of G3BP-2 in the cytoplasm of        cells (1+=low, 2+=medium, 3+=high). Unless stated otherwise,        staining was present in greater than 75% of cell population. The        column labelled nucleus indicates the presence (+) or absence        (−) of G3BP-2 in the nucleus of cancer cells.

Table 3 summarises of G3BP-1 in 24 breast tumour sections. DCIS=ductalcarcinoma in situ, IDC=infiltrating ductal carcinoma, LCIS=lobularcarcinoma in situ, ILC=infiltrating lobular carcinoma. Grade is assignedaccording to a range of factors—a well-differentiated tumour isgenerally assigned a grade 1 while a poorly differentiated tumour isassigned a grade 3. ER=oestrogen receptor status. Node status refers tothe presence (+) or absence (−) of tumour in the lymph nodes. NG=notgraded. ND=not determined. The column labelled cytoplasm indicates thelevel of expression of G3BP-1 in the cytoplasm of cells (1+=low,2+=medium, 3+=high). Unless stated otherwise, staining was present ingreater than 75% of cell population. The column labelled nucleusindicates the presence (+) or absence (−) of G3BP-1 in the nucleus ofcells.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave a meaning as commonly understood by those of ordinary skill in theart to which the invention belongs. Although any method and materialsimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, preferred methods andmaterials are described. For the purpose of the present invention, thefollowing terms are defined below.

For the purposes of this invention, by “isolated” is meant material thathas been removed from its natural state or otherwise been subjected tohuman manipulation. Isolated material may be substantially oressentially free from components that normally accompany it in itsnatural state, or may be manipulated so as to be in an artificial statetogether with components that normally accompany it in its naturalstate. Isolated material includes material in native and recombinantform. For example, G3BP-2 nucleic acids and encoded polypeptides(inclusive of HsaG3BP-2a, HsaG3BP-2b isolated from human; and MmuG3BP-2aand MmuG3BP-2b isolated from mouse) have been respectively isolated fromhuman and mouse.

By “endogenous” nucleic acid or polypeptide is meant a nucleic acid orpolypeptide which may be found in a native cell, tissue or animal inisolation or otherwise.

Polypeptide or Protein

By “polypeptide” is also meant “protein”, either term referring to anamino acid polymer, comprising natural and/or non-natural amino acids asare well understood in the art. For example, G3BP-2 may be referred toas both a protein or polypeptide. “Protein” may refer to a peptide,polypeptide, or fragments thereof. “G3BP-2” protein encompasses isoformsthereof, including G3BP-2a and G3BP-2b and all other isoforms, unless aspecific isoform is referred to.

A “peptide” is a protein having no more than fifty (50) amino acids.

In one embodiment, a “fragment” includes an amino acid sequence whichconstitutes less than 100%, but at least 20%, preferably at least 30%,more preferably at least 80% or even more preferably at least 90% ofsaid polypeptide.

The fragment may also include a “biologically active fragment” whichretains biological activity of a given polypeptide or peptide. Forexample, a biologically active fragment of G3BP-2 comprises a NTF2-likefragment which is associated with binding a SH3 domain. The NTF2-likedomain includes amino acid residues 1 to 146 as shown in FIG. 1. It isunderstood that the fragment may be derived from either a native or arecombinant polypeptide or peptide. The biologically active fragmentconstitutes at least greater than 1% of the biological activity of theentire polypeptide or peptide, preferably at least greater than 10%biological activity, more preferably at least greater than 25%biological activity and even more preferably at least greater than 50%biological activity.

In another embodiment, a “fragment” is a small peptide, for example ofat least 6, preferably at least 10 and more preferably at least 20 aminoacids in length, which comprises one or more antigenic determinants orepitopes. Larger fragments comprising more than one peptide are alsocontemplated, and may be obtained through the application of standardrecombinant nucleic acid techniques or synthesized using conventionalliquid or solid phase synthesis techniques. For example, reference maybe made to solution synthesis or solid phase synthesis as described, forexample, in Chapter 9 entitled “Peptide Synthesis” by Atherton andShephard which is included in a publication entitled “SyntheticVaccines” edited by Nicholson and published by Blackwell ScientificPublications. Alternatively, peptides can be produced by digestion of apolypeptide of the invention with a suitable proteinases. The digestedfragments can be purified by, for example, high performance liquidchromatographic (HPLC) techniques.

In one form, the invention provides a protein that comprises anNTF2-like domain. The term “comprises” refers to the protein at leasthaving the NTF2-like domain and any additional amino acid sequence.

In another form, the invention provides a protein that consistsessentially of the NTF2-like domain. The term “consists essentially of”in relation to a protein refers to a protein that in addition to thestated portion thereof, eg. the NTF2-like domain, consists of no morethan 30 additional amino acids located the amino and/or carboxylterminal end(s) thereof. Preferably, the protein consists of no morethan 20 additional amino acids. More preferably, the protein consists ofbetween 1-10 additional amino acids. The additional amino acids or“additions” may comprise a fusion protein, for example those well knownin the art including GST and (6×-HIS)-tag as described hereinafter.

In another form, the invention provides a protein that “consist of” theNTF2-like domain. This means a protein comprising an amino acid sequenceof only the NTF2-like domain.

The NTF2-like domain is set forth in SEQ ID NO: 5, referring to aminoacid residues 1 to 146, wherein amino acid residue 1 is the firstmethionine (M).

As used herein, “variant” polypeptides are polypeptides of the inventionin which one or more amino acids have been replaced by different aminoacids. It is well understood in the art that some amino acids may bechanged to others with broadly similar properties without changing thenature of the activity of the polypeptide (conservative substitutions).Exemplary conservative substitutions in the polypeptide may be madeaccording to Table 1.

Substantial changes in function are made by selecting substitutions thatare less conservative than those shown in Table 1. Other replacementswould be non-conservative substitutions and relatively fewer of thesemay be tolerated. Generally, the substitutions which are likely toproduce the greatest changes in a polypeptide's properties are those inwhich (a) a hydrophilic residue (e.g., Ser or Thr) is substituted for,or by, a hydrophobic residue (e.g. Leu, Ile, Phe or Val); (b) a cysteineor proline is substituted for, or by, any other residue; (c) a residuehaving an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp) or(d) a residue having a bulky side chain (e.g., Phe or Trp) issubstituted for, or by, one having a smaller side chain (e.g., Ala, Ser)or no side chain (e.g., Gly).

Polypeptide and Nucleic Acid Sequence Comparison

Terms used herein to describe sequence relationships between respectivenucleic acids and polypeptides include “comparison window”, “sequenceidentity”, “percentage of sequence identity” and “substantial identity”.Because respective nucleic acids/polypeptides may each comprise (1) onlyone or more portions of a complete nucleic acid/polypeptide sequencethat are shared by the nucleic acids/polypeptides, and (2) one, or moreportions which are divergent between the nucleic acids/polypeptides,sequence comparisons are typically performed by comparing sequences overa “comparison window” to identify and compare local regions of sequencesimilarity. A “comparison window” refers to a conceptual segment oftypically at least 6 contiguous residues that is compared to a referencesequence. The comparison window may comprise additions or deletions(i.e., gaps) of about 20% or less as compared to the reference sequence(which does not comprise additions or deletions) for optimal alignmentof the respective sequences. Optimal alignment of sequences for aligninga comparison window may be conducted by computerised implementations ofalgorithms (for example ECLUSTALW and BESTFIT provided by WebAngis GCG,2D Angis, GCG and GeneDoc programs, incorporated herein by reference) orby inspection and the best alignment (i.e., resulting in the highestpercentage homology over the comparison window) generated by any of thevarious methods selected.

The ECLUSTALW program is used to align multiple sequences. This programcalculates a multiple alignment of nucleotide or amino acid sequencesaccording to a method by Thompson, J. D., Higgins, D. G. and Gibson, T.J. (1994). This is part of the original ClustalW distribution, modifiedfor inclusion in EGCG. The BESTFIT program aligns forward and reversesequences and sequence repeats. This program makes an optimal alignmentof a best segment of similarity between two sequences. Optimalalignments are determined by inserting gaps to maximize the number ofmatches using the local homology algorithm of Smith and Waterman.ECLUSTALW and BESTFIT alignment packages are offered in WebANGIS GCG(The Australian Genomic Information Centre, Building JO3, The Universityof Sydney, N.S.W 2006, Australia).

Reference also may be made to the BLAST family of programs as forexample disclosed by Altschul et al., 1997, Nucl. Acids Res. 25 3389,which is incorporated herein by reference.

A detailed discussion of sequence analysis can be found in Chapter 19.3of Ausubel et al, supra.

The term “sequence identity” is used herein in its broadest sense toinclude the number of exact nucleotide or amino acid matches havingregard to an appropriate alignment using a standard algorithm, havingregard to the extent that sequences are identical over a window ofcomparison. Thus, a “percentage of sequence identity” is calculated bycomparing two optimally aligned sequences over the window of comparison,determining the number of positions at which the identical nucleic acidbase (e.g., A, T, C, G, U) occurs in both sequences to yield the numberof matched positions, dividing the number of matched positions by thetotal number of positions in the window of comparison (i.e., the windowsize), and multiplying the result by 100 to yield the percentage ofsequence identity. For example, “sequence identity” may be understood tomean the “match percentage” calculated by the DNASIS computer program(Version 2.5 for windows; available from Hitachi Software engineeringCo., Ltd., South San Francisco, Calif., USA).

As generally used herein, a “homolog” shares a definable nucleotide oramino acid sequence relationship with a nucleic acid or polypeptide ofthe invention as the case may be.

“Polypeptide homologs” share at least 80%, preferably at least 90% andmore preferably at least 95% sequence identity with the amino acidsequences of polypeptides of the invention as hereinbefore described.Polypeptide homologs include, for example G3BP-1. Also included areG3BP-2 isoforms G3BP-2a and G3BP-2b.

Included within the scope of homologs are “orthologs”, which arefunctionally-related polypeptides and their encoding nucleic acids,isolated from other organisms. For example G3BP-2 polypeptides isolatedfrom human (eg. HsaG3BP-2a, HsaG3BP-2b) and mouse (eg. MmuG3BP-2a,MmuG3BP-2b).

With regard to polypeptide variants, these can be created bymutagenising a polypeptide or by mutagenising an encoding nucleic acid,such as by random mutagenesis or site-directed mutagenesis. Examples ofnucleic acid mutagenesis methods are provided in Chapter 9 of CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et al., supra which isincorporated herein by reference.

It will be appreciated by the skilled person that site-directedmutagenesis is best performed where knowledge of the amino acid residuesthat contribute to biological activity is available. In many cases, thisinformation is not available, or can only be inferred by molecularmodeling approximations, for example.

In such cases, random mutagenesis is contemplated. Random mutagenesismethods include chemical modification of proteins by hydroxylamine (Ruanet al., 1997, Gene 188 35), incorporation of dNTP analogs into nucleicacids (Zaccolo et al., 1996, J. Mol. Biol. 255 589) and PCR-based randommutagenesis such as described in Stemmer, 1994, Proc. Natl. Acad. Sci.USA 91 10747 or Shafikhani et al., 1997, Biotechniques 23 304, each ofwhich references is incorporated herein. It is also noted that PCR-basedrandom mutagenesis kits are commercially available, such as theDiversify™ kit (Clontech).

As used herein, “derivative” polypeptides are polypeptides of theinvention which have been altered, for example by conjugation orcomplexing with other chemical moieties or by post-translationalmodification techniques as would be understood in the art. Suchderivatives include amino acid deletions and/or additions topolypeptides of the invention, or variants thereof.

“Additions” of amino acids may include fusion of the peptide orpolypeptides or variants thereof with other peptides or polypeptides.Particular examples of such peptides include amino (N) and carboxyl (C)terminal amino acids added for use as “tags”. Use of an N-terminal6×-His tag for isolating an expressed fusion polypeptide is describedherein.

N-terminal and C-terminal tags include known amino acid sequences whichbind a specific substrate, or bind known antibodies, preferablymonoclonal antibodies. pRSET B vector (ProBond™; Invitrogen Corp.) is anexample of a vector comprising an N-terminal 6×-His-tag which bindsProBond™ resin.

Other derivatives contemplated by the invention include, modification toside chains, incorporation of unnatural amino acids and/or theirderivatives during peptide or polypeptide synthesis and the use of crosslinkers and other methods which impose conformational constraints on thepolypeptides, fragments and variants of the invention. Examples of sidechain modifications contemplated by the present invention includemodifications of amino groups such as by acylation with aceticanhydride; acylation of amino groups with succinic anhydride andtetrahydrophthalic anhydride; amidination with methylacetimidate;carbamoylation of amino groups with cyanate; pyridoxylation of lysinewith pyridoxal-5-phosphate followed by reduction with NaBH₄; reductivealkylation by reaction with an aldehyde followed by reduction withNaBH₄; and trinitrobenzylation of amino groups with2,4,6-trinitrobenzene sulphonic acid (TNBS).

The carboxyl group may be modified by carbodiimide activation viaO-acylisourea formation followed by subsequent derivitization, by way ofexample, to a corresponding amide.

The guanidine group of arginine residues may be modified by formation ofheterocyclic condensation products with reagents such as2,3-butanedione, phenylglyoxal and glyoxal.

Sulphydryl groups may be modified by methods such as performic acidoxidation to cysteic acid; formation of mercurial derivatives using4-chloromercuriphenylsulphonic acid, 4-chloromercuribenzoate;2-chloromercuri-4-nitrophenol, phenylmercury chloride, and othermercurials; formation of a mixed disulphides with other thiol compounds;reaction with maleimide, maleic anhydride or other substitutedmaleimide; carboxymethylation with iodoacetic acid or iodoacetamide; andcarbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified, for example, by alkylation of theindole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphonyl halides orby oxidation with N-bromosuccinimide.

Tyrosine residues may be modified by nitration with tetranitromethane toform a 3-nitrotyrosine derivative.

The imidazole ring of a histidine residue may be modified byN-carbethoxylation with diethylpyrocarbonate or by alkylation withiodoacetic acid derivatives.

Examples of incorporating unnatural amino acids and derivatives duringpeptide synthesis include, use of 4-amino butyric acid, 6-aminohexanoicacid, 4-amino-3-hydroxy-5-phenylpentanoic acid,4-amino-3-hydroxy-6-methylheptanoic acid, t-butylglycine, norleucine,norvaline, phenylglycine, ornithine, sarcosine, 2-thienyl alanine and/orD-isomers of amino acids.

Polypeptides in relation to the invention such as those exemplified inFIG. 1 (inclusive of fragments, variants, derivatives and homologs ingeneral) may be prepared by any suitable procedure known to those ofskill in the art.

For example, the polypeptide may be prepared by a procedure includingthe steps of:

-   -   (i) preparing an expression construct which comprises a        recombinant nucleic acid of the invention, operably linked to        one or more regulatory nucleotide sequences, for example a T7        promoter;    -   (ii) transfecting or transforming the expression construct into        a suitable host cell, for example E. coli; and    -   (iii) expressing the polypeptide in said host cell.

Preferably, the recombinant nucleic acid of the invention encodes apolypeptide as shown in FIG. 1, or fragment thereof.

Recombinant proteins may be conveniently expressed and purified by aperson skilled in the art using commercially available kits, for example“ProBond™ Purification System” available from Invitrogen Corporation,Carlsbad, Calif., USA, herein incorporated by reference. Alternatively,standard molecular biology protocols may be used, as for exampledescribed in Sambrook, et al., MOLECULAR CLONING. A Laboratory Manual(Cold Spring Harbor Press, 1989), incorporated herein by reference, inparticular Sections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGYEds. Ausubel et al., (John Wiley & Sons, Inc. 1995-1999), incorporatedherein by reference, in particular Chapters 10 and 16; and CURRENTPROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons,Inc. 1995-1999) which is incorporated by reference herein, in particularChapters 1, 5, 6 and 7.

Nucleic Acids

The term “nucleic acid” as used herein designates single or doublestranded mRNA, RNA, cRNA and DNA, said DNA inclusive of cDNA and genomicDNA.

The term “isolated nucleic acid” as used herein refers to a nucleic acidsubjected to in vitro manipulation into a form not normally found innature. Isolated nucleic acid include both native and recombinant(non-native) nucleic acids. For example, a nucleic acid isolated fromhuman or mouse.

A “polynucleotide” is a nucleic acid having eighty (80) or morecontiguous nucleotides, while an “oligonucleotide” has less than eighty(80) contiguous nucleotides.

The term “consists essentially of in relation to a nucleic acid refersto a nucleic acid having no more than 90 nucleotides located at the 5′and/or 3′ thereof. Preferably, the nucleic acid consist of no more than60 additional nucleic acids, more preferably the nucleic acid consist ofbetween 1-30 nucleotides.

A “probe” may be a single or double-stranded oligonucleotide orpolynucleotide, suitably labeled for the purpose of detectingcomplementary sequences in Northern or Southern blotting, for example.

A “primer” is usually a single-stranded oligonucleotide, preferablyhaving 20-50 contiguous nucleotides, which is capable of annealing to acomplementary nucleic acid utemplate” and being extended in atemplate-dependent fashion by the action of a DNA polymerase such as Taqpolymerase, RNA-dependent DNA polymerase or Sequenase™. For example, thefollowing primers were used for chromosomal mapping of human G3BP-1 andG3BP-2.

HsaG3BP-1 Specific Primers: 5′GGAGGCATGGTGCAGAAACCA; [SEQ ID NO:12] and5′CAGGAAAGGGAAGAGAGGGAG [SEQ ID NO:13]

HsaG3BP-2 Specific Primers: 5′GTCTTGGCAGTGGTACATTAT; [SEQ ID NO:14] and5′AGTTCACTTTGTCGTAGATAGTTTAAG [SEQ ID NO:15]

For the purposes of host cell expression, the recombinant nucleic acidis operably linked to one or more regulatory sequences in an expressionvector, for example a T7 promoter.

An “expression vector” may be either a self-replicatingextra-chromosomal vector such as a plasmid, or a vector that integratesinto a host genome. An example of an expression vector is pRSET B(Invitrogen Corp.) and derivations thereof.

By “operably linked” is meant that said regulatory nucleotidesequence(s) is/are positioned relative to the recombinant nucleic acidof the invention to initiate, regulate or otherwise controltranscription.

Regulatory nucleotide sequences will generally be appropriate for thehost cell used for expression. Numerous types of appropriate expressionvectors and suitable regulatory sequences are known in the art for avariety of host cells.

Typically, said one or more regulatory nucleotide sequences may include,promoter sequences, leader or signal sequences, ribosomal binding sites,transcriptional start and termination sequences, translational start andtermination sequences, and enhancer or activator sequences.

Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. For example, the lac promoter is inducible by IPTG.

The expression vector may further comprise a selectable marker gene toallow the selection of transformed host cells. Selectable marker genesare well known in the art and will vary with the host cell used. Forexample, an ampicillin resistance gene for selection of positivelytransformed host cells when grown in a medium comprising ampicillin.

The expression vector may also include a fusion partner (typicallyprovided by the expression vector) so that the recombinant polypeptideof the invention is expressed as a fusion polypeptide with the fusionpartner. An advantage of fusion partners is that they assistidentification and/or purification of the fusion polypeptide.Identification and/or purification may include using a monoclonalantibody or substrate specific for the fusion partner, for example a6×-His tag or GST. A fusion partner may also comprise a leader sequencefor directing secretion of a recombinant polypeptide, for example analpha-factor leader sequence.

Well known examples of fusion partners include hexahistidine(6×-HIS)-tag, N-Flag, Fc portion of human IgG, glutathione-5-transferase(GST) and maltose binding protein (MBP), which are particularly usefulfor isolation of the fusion polypeptide by affinity chromatography. Forthe purposes of fusion polypeptide purification by affinitychromatography, relevant matrices for affinity chromatography mayinclude nickel-conjugated or cobalt-conjugated resins, fusionpolypeptide specific antibodies, qlutathione-conjugated resins, andamylose-conjugated resins respectively. Some matrices are available in“kit” form, such as the ProBond™ Purification System (Invitrogene Corp.)which incorporates a 6×-His fusion vector and purification usingProBond™ resin.

In order to express the fusion polypeptide, it is necessary to ligate anucleic acid according to the invention into the expression vector sothat the translational reading frames of the fusion partner and thenucleotide sequence of the invention coincide.

The fusion partners may also have protease cleavage sites, for exampleenterokinase (available from Invitrogen Corp. as EnterokinaseMax™),Factor X_(a) or Thrombin, which allow the relevant protease to digestthe fusion polypeptide of the invention and thereby liberate therecombinant polypeptide of the invention therefrom. The liberatedpolypeptide can then be isolated from the fusion partner by subsequentchromatographic separation.

Fusion partners may also include within their scope “epitope tags”,which are usually short peptide sequences for which a specific antibodyis available.

As hereinbefore, polypeptides of the invention may be produced byculturing a host cell transformed with an expression constructcomprising a nucleic acid encoding a polypeptide, or polypeptidehomolog, of the invention. The conditions appropriate for polypeptideexpression will vary with the choice of expression vector and the hostcell. For example, a nucleotide sequence of the invention may bemodified for successful or improved polypeptide expression in a givenhost cell. Modifications include altering nucleotides depending onpreferred codon usage of the host cell. Alternatively, or in addition, anucleotide sequence of the invention may be modified to accommodate hostspecific splice sites or lack thereof. These modifications may beascertained by one skilled in the art.

Host cells for expression may be prokaryotic or eukaryotic.

Useful prokaryotic host cells are bacteria.

A typical bacteria host cell is a strain of E. coli.

Useful eukaryotic cells are yeast, SF9 cells that may be used with abaculovirus expression system as described herein, and other mammaliancells.

The recombinant polypeptide may be conveniently prepared by a personskilled in the art using standard protocols as for example described inSambrook, et al., MOLECULAR CLONING. A Laboratory Manual (Cold SpringHarbor Press, 1989), incorporated herein by reference, in particularSections 16 and 17; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubelet al., (John Wiley & Sons, Inc. 1995-1999), incorporated herein byreference, in particular Chapters 10 and 16; and CURRENT PROTOCOLS INPROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons, Inc. 1995-1999)which is incorporated by reference herein, in particular Chapters 1, 5and 6.

In one embodiment, nucleic acid homologs encode polypeptide homologs ofthe invention, inclusive of variants, fragments and derivatives thereof.

In another embodiment, nucleic acid homologs share at least 60%,preferably at least 70%, more preferably at least 80%, and even morepreferably at least 90% sequence identity with the nucleic acids of theinvention.

In yet another embodiment, nucleic acid homologs hybridise to nucleicacids of the invention under at least low stringency conditions,preferably under at least medium stringency conditions and morepreferably under high stringency conditions.

“Hybridise and Hybridisation” is used herein to denote the pairing of atleast partly complementary nucleotide sequences to produce a DNA-DNA,RNA-RNA or DNA-RNA hybrid. Hybrid sequences comprising complementarynucleotide sequences occur through base-pairing.

In DNA, complementary bases are:

-   -   (i) A and T; and    -   (ii) C and G.

In RNA, complementary bases are:

-   -   (i) A and U; and    -   (ii) C and G.

In RNA-DNA hybrids, complementary bases are:

-   -   (i) A and U;    -   (ii) A and T; and    -   (iii) G and C.

Modified purines (for example, inosine, methylinosine andmethyladenosine) and modified pyrimidines (thiouridine andmethylcytosine) may also engage in base pairing.

“Stringency” as used herein, refers to temperature and ionic strengthconditions, and presence or absence of certain organic solvents and/ordetergents during hybridisation. The higher the stringency, the higherwill be the required level of complementarity between hybridizingnucleotide sequences.

“Stringent conditions” designates those conditions under which onlynucleic acid having a high frequency of complementary bases willhybridize.

Reference herein to low stringency conditions includes and encompasses:—

-   -   (i) from at least about 1% v/v to at least about 15% v/v        formamide and from at least about 1 M to at least about 2 M salt        for hybridisation at 42° C., and at least about 1 M to at least        about 2 M salt for washing at 42° C.; and    -   (ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH        7.2), 7% SDS for hybridization at 65° C., and (i) 2×SSC, 0.1%        SDS; or (ii) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS        for washing at room temperature.

Medium stringency conditions include and encompass:—

-   -   (i) from at least about 16% v/v to at least about 30% V/v        formamide and from at least about 0.5 M to at least about 0.9 M        salt for hybridisation at 42° C., and at least about 0.5 M to at        least about 0.9 M salt for washing at 42° C.; and    -   (ii) 1% Bovine Serum Albumin (BSA), 1 mM EDTA, 0.5 M NaHPO₄ (pH        7.2), 7% SDS for hybridization at 65° C. and (a) 2×SSC, 0.1%        SDS; or (b) 0.5% BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 5% SDS        for washing at 42° C.

High stringency conditions include and encompass:—

-   -   (i) from at least about 31% v/v to at least about 50% v/v        formamide and from at least about 0.01 M to at least about 0.15        M salt for hybridisation at 42° C., and at least about 0.01 M to        at least about 0.15 M salt for washing at 42° C.;    -   (ii) 1% BSA, 1 mM EDTA, 0.5 M NaHPO₄ (pH 7.2), 7% SDS for        hybridization at 65° C., and (a) 0.1×SSC, 0.1% SDS; or (b) 0.5%        BSA, 1 mM EDTA, 40 mM NaHPO₄ (pH 7.2), 1% SDS for washing at a        temperature in excess of 65° C. for about one hour; and    -   (iii) 0.2×SSC, 0.1% SDS for washing at or above 68° C. for about        20 minutes.

In general, the T_(m) of a duplex DNA decreases by about 1° C. withevery increase of 1% in the number of mismatched bases.

Notwithstanding the above, stringent conditions are well known in theart, such as described in Chapters 2.9 and 2.10 of Ausubel et al.,supra, which are herein incorporated by reference. A skilled addresseewill also recognize that various factors can be manipulated to optimizethe specificity of the hybridization. Optimization of the stringency ofthe final washes can serve to ensure a high degree of hybridization.

Typically, complementary nucleotide sequences are identified by blottingtechniques that include a step whereby nucleotides are immobilized on amatrix (preferably a synthetic membrane such as nitrocellulose), ahybridization step, and a detection step. Southern blotting is used toidentify a complementary DNA sequence; Northern blotting is used toidentify a complementary RNA sequence. Dot blotting and slot blottingcan be used to identify complementary DNA/DNA, DNA/RNA or RNA/RNApolynucleotide sequences. Such techniques are well known by thoseskilled in the art, and have been described in Ausubel et al., supra, atpages 2.9.1 through 2.9.20, herein incorporated by reference.

According to such methods, Southern blotting involves separating DNAmolecules according to size by gel electrophoresis, transferring thesize-separated DNA to a synthetic membrane, and hybridizing the membranebound DNA to a complementary nucleotide sequence.

In dot blotting and slot blotting, DNA samples are directly applied to asynthetic membrane prior to hybridization as above.

An alternative blotting step is used when identifying complementarynucleic acids in a cDNA or genomic DNA library, such as through theprocess of plaque or colony hybridisation. Other typical examples ofthis procedure are described in Chapters 8-12 of Sambrook et al., suprawhich are herein incorporated by reference.

Typically, the following general procedure can be used to determinehybridisation conditions. Nucleic acids are blotted/transferred to asynthetic membrane, as described above. A nucleotide sequence of theinvention is labeled as described above, and the ability of this labelednucleic acid to hybridise with an immobilized nucleotide sequenceanalysed.

A skilled addressee will recognise that a number of factors influencehybridisation. The specific activity of radioactively labeledpolynucleotide sequence should typically be greater than or equal toabout 10⁸ dpm/μg to provide a detectable signal. A radiolabelednucleotide sequence of specific activity 10⁸ to 10⁹ dpm/μg can detectapproximately 0.5 pg of DNA. It is well known in the art that sufficientDNA must be immobilised on the membrane to permit detection. It isdesirable to have excess immobilised DNA, usually 10 pg. Adding an inertpolymer such as 10% (w/v) dextran sulfate (MW 500,000) or polyethyleneglycol 6000 during hybridisation can also increase the sensitivity ofhybridisation (see Ausubel et al., supra at 2.10.10).

To achieve meaningful results from hybridisation between a nucleic acidimmobilised on a membrane and a labeled nucleic acid, a sufficientamount of the labeled nucleic acid must be hybridised to the immobilisednucleic acid following washing. Washing ensures that the labeled nucleicacid is hybridised only to the immobilised nucleic acid with a desireddegree of complementarity to the labeled nucleic acid.

Methods for detecting labeled nucleic acids hybridised to an immobilisednucleic acid are well known to practitioners in the art. Such methodsinclude autoradiography, chemiluminescent, fluorescent and colourimetricdetection.

Nucleic acid homologs of the invention may be prepared according to thefollowing procedure:

-   -   (i) obtaining a nucleic acid extract from a suitable host, for        example a bacterial species;    -   (ii) creating primers which are optionally degenerate wherein        each comprises a portion of a nucleotide sequence of the        invention; and    -   (iii) using said primers to amplify, via nucleic acid        amplification techniques, one or more amplification products        from said nucleic acid extract.

As used herein, an “amplification product” refers to a nucleic acidproduct generated by nucleic acid amplification techniques.

Suitable nucleic acid amplification techniques are well known to theskilled addressee, and include PCR as for example described in Chapter15 of Ausubel et al. supra, which is incorporated herein by reference;strand displacement amplification (SDA) as for example described in U.S.Pat. No. 5,422,252 which is incorporated herein by reference; rollingcircle replication (RCR) as for example described in Liu et al., 1996,J. Am. Chem. Soc. 118 1587 and International application WO 92/01813;and Lizardi and Caplan, International Application WO 97/19193, which areincorporated herein by reference; nucleic acid sequence-basedamplification (NASBA) as for example described by Sooknanan et al.,1994, Biotechniques 17 1077, which is incorporated herein by reference;ligase chain reaction (LCR) as for example described in InternationalApplication WO89/09385 which is incorporated herein by reference; andQ-β replicase amplification as for example described by Tyagi et al.,1996, Proc. Natl. Acad. Sci. USA 93 5395 which is incorporated herein byreference. Preferably, amplification is by PCR using primers disclosedherein.

G3BP-2 and Breast Cancer

G3BP-2 protein was found to be in breast cancers that are derived fromepithelial tissues and non-proliferative tissues. This was an unexpectedresult, at least partly because it was not known that expression wouldbe shown to be specific to tumours and that up-regulation of G3BP-2would occur so early in the development of the tumour. G3BP-2 isup-regulated in approximately 80% of breast cancers studied. Thiscompares to genes that are well recognised as causing breast cancerssuch as Brca1, which is found to be mutated in 15% of familial breastcancers (percentages depend on the country of the study). Familialbreast cancers only represent 15-30% of total breast cancers, whichmeans that Brca1 is causative of only 2-5% of all breast cancers.

The inventors have proposed a model that suggests G3BP-2 migrates to thenucleus of the cell to pick up transcripts and export them to theribosome so that translation of selected gene transcripts can beregulated at the level of the ribosome. The inventors have proposed thismodel because of a surprising finding that G3BP-2 can beimmunoprecipitated with ribosomal proteins normally associated withpolysomes. This suggests that a possible method of action may be toexport the transcripts of oncogenes that regulate cell cycle to theribosome to up-regulate their transcription and thereby enhancing cancerprogression. Interestingly it also suggests that G3BP-2 may represent aconnection between signal transduction and RNA-processing.

G3BP-2 may not actually be causative of breast cancer, but may berequired by the cancer to cause proliferation and thereby account forits up-regulation in 80% of breast cancers. G3BP-2 is specificallylocalised to sub-cellular compartments in a cell cycle dependent manner,moving into the nucleus during proliferation and then back out to thecytoplasm. G3BP-2 appears to “short-circuit” normal ras-GAP¹²⁰signalling by receiving messages directly from GAP¹²⁰ and moving intothe cell nucleus. In the nucleus G3BP-2 is most likely binding withtranscript targets (eg. c-myc) and exporting them from the nucleus tothe ribosomes in the cytoplasm. The inventors have co-immunoprecipitatedG3BP-2 with a series of polypeptides normally associated withtranslational active ribosomes. Accordingly, G3BP-2 may be facilitatingincreased proliferation of breast cancers by allowing the up-regulationof oncogenic gene transcripts (eg. c-myc). Therapeutics designed toblock the activity of G3BP-2, in particular at the N-terminal NTF2-likedomain, may limit cancer progression.

Cloning, Sequence Homology and Structural Homology

The inventors previously reported cloning MmuG3BP-2 (MMU65313) in ageneral PCR-based screening for RRM-containing proteins (Kennedy et al.1997). This was achieved by using degenerate primers designed toconsensus sequences within the RRM (Birney et al., 1993) and using theamplified PCR product to screen a late-primitive-streak stage mouseembryo cDNA library to isolate a full-length cDNA. Due to the conservedsequence homology between the G3BP genes, the coding region of theMmuG3BP-2 cDNA can be used as a useful tool to recognise both humanG3BP-2 and MmuG3BP-1 in Northern analysis and library screening. Theinventors used the coding region of MmuG3BP-2 to isolate and cloneMmuG3BP-1 (MMAB1927) and human G3BP-2 (HsaG3BP-2) from thelate-primitive-streak stage mouse embryo cDNA library and a foetal humanbrain cDNA library respectively. The clones were sequenced and analysedto identify Met start codons, open reading frames and stop codons.Protein sequence comparison (FIG. 3) between HsaG3BP-2 and MmuG3BP-2show 98.5% identity, HsaG3BP-2 and HsaG3BP-1 (HS3251910) show 65%identity and HsaG3BP-1 shares 94.4% sequence identity with MmuG3BP-1. InFIG. 3, amino acids are shown in single letter format and grouped inblocks of 10. Numbering at the end of the line indicates the amino acidposition within the indicated protein. Dashes within the alignedsequences indicate conserved amino acids with respect to HsaG3BP-2a,amino acid changes between proteins have been indicated by theappropriate substitution. Spaces within the aligned proteins indicategaps inserted into the sequences to maintain co-linearity. Boxesrepresent proline rich sequences (PxxP). Sequences in italics indicatethe acid-rich domain. Ovals represent components of an RGG domain, notethat the RGG domains of G3BP-1 and G3BP-2 are divergent. Underlined anddouble underlined sequences indicate RNP-2 and RNP-1 respectively.

Screening of the libraries also revealed that at least two differentG3BP-2 isoforms are produced in both mouse and human. The alternativesplicing event, which is 100% conserved between mouse and human, deletes99 nucleotides from the coding region and does not introduce any stopcodons nor a frame shift. Both these transcripts are translated in vivoas was confirmed by Western blot analysis and recombinant proteinexpression (see below). The longer isoform has been designated G3BP-2a(human G3BP-2a accession number AF145285, mouse G3BP-2a accession numberAF145285). The shorter protein isoform is referred to as G3BP-2b (humanG3BP-2b accession number AF053535 and AF051311 and mouse G3BP-2baccession number MMU65313) differs from G3BP-2a by a 33 amino aciddeletion from the central region of the primary structure (FIG. 3). Athird MmuG3BP-2 transcript was detected and cloned (referred to hereinas G3BP-2c); however, sequence analysis indicated that translation ofthis transcript would lead to a truncated gene product and so far nocorresponding protein has been detected in cells or tissues suggestingthat it may not be translated.

All G3BPs share highly conserved RNP-1 and RNP-2 sequences, which areconsensus motifs of RRMs. The most notable difference between G3BP-2 andG3BP-1 RRMs is a Val to Ile substitution in the RNP-2 consensus sequence(FIG. 3). The structure of the G3BP RRM has been reported elsewhere(Kennedy et al. 1997). In addition, the G3BPs contain a conservedacid-rich domain and an RGG domain (Birney et al. 1993), both of whichare commonly found in RNA-binding proteins. It should be noted thatthere are considerable differences in the RGG domain structure betweenG3BP-1 and G3BP-2 (see FIG. 3) and this may result in a different RNAtarget specificity. Although acid-rich domains are found in associationwith a variety of RNA-binding proteins such as hnRNP C and nucleolin,the function of this domain remains unclear and maybe involved inprotein-protein interactions. The most significant difference betweenthe G3BP-1 and G3BP-2 proteins lies in the number of potential SH3domain-binding motifs (PxxP, where x represents any amino acid) (Lee etal. 1996). The G3BP-2a protein comprises a cluster of four PxxP motifsbetween the acid-rich and RRM domains whereas G3BP-2b contains five inthe homologous region (FIG. 3). The additional proline-rich PxxP motifin G3BP-2b is generated by the 33 amino acids spliced out of G3BP-2a. Incontrast to the multiple PxxP clusters found in G3BP-2s, G3BP-1 containsonly one such motif in the homologous region of the protein (FIG. 3).Furthermore, both G3BP-1 and G3BP-2 comprise a conserved PxxP motif(PGGP) within their non-conserved RGG domains (FIG. 3). The specificconservation of the minimal SH3 domain-binding motif within a region ofthe protein, which is generally not conserved, may suggest a retainedfunction although this remains to be determined. The overall differencesin the number of potential SH3 domain-binding motifs between the G3BPsmay indicate that in vivo they may bind different SH3 domain-containingpartners or have different affinities for the same protein.

Protein Expression in Insect Cells

MmuG3BP-2a and 2b cDNAs encode proteins whose predicted sizes are 58.2kDa and 52 kDa respectively. The expressed recombinant proteins haveapparent molecular weights of 68.5 kDa and 62 kDa respectively, asdetermined by SDS-PAGE. These differences in predicted and apparentmasses are consistent with an increase in mass due to post translationalmodifications and are similar to those reported for HsaG3BP-1 (predictedmass of 52, observed apparent mass of 68 kDa) (Parker et al. 1996).

Mapping of G3BP-rasGAP¹²⁰ Interactions

Interactions between G3BP-1 and rasGAP¹²⁰ have been reported and showthat G3BP-1 specifically interacts with the SH3 domain of rasGAP¹²⁰(Parker et al. 1996) implicating G3BP in the rasGAP¹²⁰ signaltransduction pathway (see also Pazman et al. 2000). However, the regionwithin the G3BPs responsible for the observed interaction with rasGAP¹²⁰has not been thoroughly investigated. Initially it was presumed that theinteractions would be facilitated through proline rich motifs that areknown to interact with SH3 domains (Lee et al. 1996). To further map theinteractions between G3BPs and the SH3 domain of rasGAP¹²⁰ severalGST-G3BP peptide fusions were expressed (FIG. 4) and probed in beadbinding assays with His-tagged N-terminal rasGAP¹²⁰ peptides. The G3BPpeptide constructs were designed to represent truncated proteinscontaining single or multiple domains as well as peptides that wouldcontain isolated proline rich motifs (FIG. 4). In the assays GST-G3BPpeptides are bound to glutathione beads and His-tagged N-terminalrasGAP¹²⁰ is added to the different constructs. Specific protein-proteininteractions between the G3BP peptides and the SH3 domain of rasGAP¹²⁰are detected by running the bound proteins on a Western blot andidentifying His-tagged rasGAP¹²⁰ by probing with an anti-His antibody.The results show (FIG. 4) that the interaction between the SH3 domain ofrasGAP¹²⁰ maps to the NTF2-like domain contained within the N-terminaldomain of the G3BPs but not the proline rich motifs. No interactionswere detected with the other domains of G3BP including the Acid-Richdomain, the RRM or the RGG-rich domain (FIG. 4).

FIG. 4A shows a schematic representation of sub-domains and motifscontained within G3BP-2a and G3BP-1 proteins and includes the N-terminalNTF2-like, Acid-rich, RGG and proline-rich domains as well as theRNA-recognition motif (see insert for details). Below the respectivefull length proteins are shown various truncated peptides that wereexpressed as GST-fusion proteins to map interactions with the N-terminalSH3 domain containing region of rasGAP²⁰. The numbering corresponds tothe amino acid at the site of the peptide truncation (ie. δ-2a-N146represents the truncated G3BP-2a peptide including the N-terminal aminoacids 1 to 146), the relative position of these truncations is shown toapproximate scale in the full length proteins. FIGS. 4B and 4C showWestern blot analysis of Glutathione beads bound with various GST-G3BPpeptides and probed with the His-tagged N-terminal SH3 domain containingregion of rasGAP¹²⁰.

G3BP-rasGAP¹²⁰ interactions were determined by probing the Western blotswith anti-His antibodies. The results show that the N-terminal SH3domain containing region of rasGAP¹²⁰ interacted with the NTF2 likedomain of G3BP-2a (δ-2a-N146, panel B) and any peptide of G3BP-2a thatcomprised the NTF2 like domain (δ-2a-N205, δ-2a-N257, δ-2a-N329 and fulllength G3BP-2a, panel B). The results obtained from G3BP-1 areconsistent with these results from G3BP-2a and show that full lengthG3BP-1, δ-1-N229 and δ-1-N309 interact with the N-terminal SH3 domaincontaining region of rasGAP¹²⁰. Truncated peptides of either G3BP-2a orG3BP-1 that did not contain an intact NTF2-like domain failed to bindwith the SH3 region of rasGAP¹²⁰.

The results reported herein were confirmed using far-Western protocols(data not shown) and are consistent with the data obtained from the beadbinding assays.

G3BPs have a Tissue Specific Expression

Antibodies raised against isoform specific synthetic peptides determinedfrom G3BP-1 and G3BP-2 sequences were used to probe western blots oftotal cell lysates from adult mouse tissues (FIG. 5). FIG. 5, panel Ashows tissues probed with anti-G3BP-1 polyclonal antibodies whereaspanel B shows a collage of tissues probed with anti-G3BP-2 polyclonalantibodies. Some tissues are shown to express both isoforms of G3BP-2(FIG. 5, panel B) including lung, liver, kidney, stomach and colon (alsopancreas and testis, data not shown). Other tissues are restricted toonly expressing G3BP-2a (upper band in FIG. 5, panel B) including brain,muscle (small amount of G3BP-2b expression is seen and is presumablycaused by the presence of different cell populations within the sample),and heart. Small intestine expresses only MmuG3BP-2b (lower band FIG. 5,panel B) and spleen does not express either protein at detectablelevels. Although the general expression of G3BP-1 appears lower thanthat of G3BP-2 some tissues express abundant levels of G3BP-1 andinclude lung, kidney and colon. Heart, liver and spleen also express lowlevels of G3BP-1.

FIG. 6 shows immunohistochemistry results of adult mouse tissues probedwith anti-G3BP-1 and anti-G3BP-2 antibodies. Panels A to E are probedwith an anti-G3BP-1 antibody whereas panels F to J are probed with ananti-G3BP-2 antibody. All tissue staining was visualised with horseradish peroxidase and sections were counterstained with haematoxylin.Panels A and F are brain (Ne denotes a neurone, GI denotes a glialcell), B and G are kidney (Gm denotes a glomerulus, Tu denotes atubule), C and H are colon (Ig denotes an intestinal gland) D and I aresmall intestine (V denotes a villi) and E and J are stomach (Ep denotesepithelial mucus secreting cells and Pg denotes a pyloric gland). Allphotographs are taken at 100× magnification (bars represent 100 μm) withthe exception of stomach (panels E and J), which is displayed at 50×magnification (bars represent 100 μm).

Chromosomal Location of G3BPs

Sequence data obtained from the library clones was used to design G3BP-1and G3BP-2 specific primers, which were subsequently used on theGeneBridge hybridisation panel to determine the chromosomal localisationof these genes. G3BP-1 mapped to chromosome 5 at 1.51 cR from FB25D10(lod>3.0) which places the gene between 5q33.1-5q33.3. G3BP-2 mapped tochromosome 4 at 3.36 cR from WI-5565 (lod>3.0) which places the genebetween 4q12-4q24. A plasmid artificial chromosome (PAC) library (kindlydonated by Dr. P. Ioannou, The Murdoch Institute, Australia) wassubsequently screened and isolated clones used to perform fluorescentin-situ hybridisation. The results of the FISH confirmed the geneticlocation of these genes. In addition to screening the GeneBridgehybridisation panel and the FISH analysis, the human genome sequences inthe NCBI databases (http://ncbi.nlm.nih.gov/genome/seq/) were BLASTsearched with the cDNA sequences of human G3BP-1 and G3BP-2. The resultsindicated that there are several chromosomes with matches to eitherG3BP-1 or G3BP-2, which may represent gene duplications or pseudo-genes.The BLAST analysis on its own was not sufficient to map the G3BPs,however, in conjunction with the FISH and the GeneGridge hybridisationpanel results the chromosomal localisation of these genes as indicatedabove has been confirmed.

A search of the genomic databases (available athttp://gdbwww.gdb.org/gdbreports/GeneByChromosome.4.alpha.html andhttp://gdbwww.gdb.org/gdbreports/GeneByChromosome.5.alpha.html) did notreveal any candidate loci for diseases that may represent geneticdefects/polymorphisms in this family of proteins although, there doesappear to be some clustering of RNA-binding protein genes on chromosome5q including, hnRNP A/B (GDB:128837), hnRNP H1 (GDB:5428597), ribosomalprotein L7 pseudo gene (GDB:277889), ribosomal protein S14 (GDB:119572),ribosomal protein S17a-like 1 (GDB:119573), ribosomal protein S20A(GDB:119575) and ribosomal protein S20B (GDB:119576). Chromosome 4q alsocontains several RNA-binding proteins including EIF4EL1 (GDB:126371),G-rich RNA sequence binding factor 1 (GDB:696354), hnRNP D(GDB:9391694), RNA polymerase II polypeptide B (GDB:135034) andribosomal protein L34 (GDB:9863242). Segment deletions and breakpointanalysis of regions overlapping the chromosomal location of G3BP-1suggest that this region may be involved in myeloid leukemias (Horriganet al., 1999) whereas similar studies for chromosome 4q suggest theregion containing the G3BP-2 gene may be involved in colorectal adenoma(Wong et al., 1999) and Hodgkin's disease (Atkin 1998). However, thereis insufficient data to suggest that any G3BPs are involved in humanpathologies that are linked to these regions of the human chromosomes.

Antibodies to G3BP-2

The invention also relates to antibodies against the isolated G3BP-2polypeptide, fragments, variants and derivatives thereof. A peptidefragment of G3BP-2 may comprise amino acid sequenceSATPPPAEPASLPQEPPKPRV [SEQ ID NO: 3], as herein described. Antibodies ofthe invention may be polyclonal or monoclonal. Well-known protocolsapplicable to antibody production, purification and use may be found,for example, in Chapter 2 of Coligan et al., CURRENT PROTOCOLS INIMMUNOLOGY (John Wiley & Sons NY, 1991-1994) and Harlow, E. & Lane, D.Antibodies: A Laboratory Manual, Cold Spring Harbor, Cold Spring HarborLaboratory, 1988, which are both herein incorporated by reference.

Generally, antibodies of the invention bind to or conjugate with apolypeptide, fragment, variant or derivative of the invention. Forexample, the antibodies may comprise polyclonal antibodies. Suchantibodies may be prepared for example by injecting a polypeptide,fragment, variant or derivative of the invention into a productionspecies, which may include mice or rabbits, to obtain polyclonalantisera. Methods of producing polyclonal antibodies are well known tothose skilled in the art. Exemplary protocols which may be used aredescribed for example in Coligan et al., CURRENT PROTOCOLS INIMMUNOLOGY, supra, and in Harlow & Lane, 1988, supra.

In lieu of the polyclonal antisera obtained in the production species,monoclonal antibodies may be produced using the standard method as forexample, described in an article by Köhler & Milstein, 1975, Nature 256,495, which is herein incorporated by reference, or by more recentmodifications thereof as for example, described in Coligan et al.,CURRENT PROTOCOLS IN IMMUNOLOGY, supra by immortalizing spleen or otherantibody producing cells derived from a production species which hasbeen inoculated with one or more of the polypeptides, fragments,variants or derivatives of the invention.

The invention also includes within its scope antibodies, which compriseFc or Fab fragments of the polyclonal or monoclonal antibodies referredto above. Alternatively, the antibodies may comprise single chain Fvantibodies (scFvs) against the peptides of the invention. Such scFvs maybe prepared, for example, in accordance with the methods describedrespectively in U.S. Pat. No. 5,091,513, European Patent No 239,400 orthe article by Winter & Milstein, 1991, Nature 349 293, which areincorporated herein by reference.

The antibodies of the invention may be used for affinity chromatographyin isolating natural or recombinant polypeptides of the invention. Forexample, reference may be made to immunoaffinity chromatographicprocedures described in Chapter 9.5 of Coligan et al., CURRENT PROTOCOLSIN IMMUNOLOGY, supra.

Antibodies may be purified from a suitable biological fluid of theanimal by ammonium sulfate fractionation, affinity purification or byother methods well known in the art. Exemplary protocols for antibodypurification are given in Sections 10.11 and 11.13 of Ausubel et al.,supra, which are herein incorporated by reference.

Immunoreactivity of the antibody against the native or parentpolypeptide may be determined by any suitable procedure such as, forexample, Western blot.

Mimetics, Agonists and Antagonists

G3BP-2 offers a unique possibility for therapeutics because of itsinteraction with oncogenic pathways and its unique features, whichappear to regulate cell cycle. Of particular interest is an N-terminalNTF2-like domain, which has a surprising host of activities:

-   (1) The NTF2-like domain appears to regulate nuclear localisation    through an interaction with the ran nuclear pore protein.-   (2) The expression of a known oncogene, NFκB, through interactions    with a Ubiquitin hydrolase, ODE1.-   (3) Signal transduction through interactions with GAP¹²⁰

The inventors have also determined that G3BP-2 receives its messagesfrom GAP¹²⁰ through the NTF2-like domain. The NTF2-like domain of G3BP-2is highly conserved to the entire NTF2 protein. NTF2 is a nuclear poreprotein that shuttles into the nucleus through energy dependentinteractions with ran. The inventors speculate that G3BP-2 shuttles inand out of the nucleus using the same mechanisms as NTF2 and to thisextent they have shown that G3BP-2 interacts with ran. The inventorshave also determined that the NTF2-like domain of G3BP-2 interacts withODE1, a ubiquitin hydrolase and that this interaction can increase thegene expression of NFκB, another protein implicated in tumourprogression.

Targeting the NTF2-like domain for anti-cancer therapeutics may inhibittumour cell proliferation. This would be achieved by blocking ability ofG3BP-2 to receive signals from GAP¹²⁰, to block its ability to shuttlemRNA transcripts from the nucleus to the cytoplasm and to block itsability to cause the up-regulation of NFκB.

The invention contemplates agents which may prevent or disrupt formationof polypeptide complexes comprising G3BP-2 and a native or endogenoustarget polypeptide. Such an agent may be a mimetic, which antagonizes ormimics one or more biological activities of G3BP-2 polypeptides, orhomologs thereof.

It will be appreciated that G3BP-2 comprises several recognisablesub-domains (an acid-rich domain, an RNA-recognition motif, anarginine-glycine rich motif and a proline-rich motif). A key to itsbiological activity as a polypeptide that can facilitate cancerprogression may lie in the G3BP-2 N-terminal NTF2-like domain.

The NTF2-like domain is considered to be preferred target for thescreening or design of potential G3BP-2 mimetics.

The term “mimetic” is used herein to refer to molecules that aredesigned to resemble particular functional regions of proteins orpeptides, and includes within its scope the terms “agonist”, “analogue”and “antagonist” as are well understood in the art.

An antagonist may be a competitive antagonist or non-competitiveantagonist.

It is contemplated that mimetics could be engineered which disrupt orprevent formation of polypeptide complexes between G3BP-2 and endogenoustarget polypeptides. A mimetic preferably disrupts or prevents formationof a complex between the NTF2-like domain of G3BP-2 and an endogenoustarget peptide, for example rasGAP¹²⁰. In particular, a polypeptidefragment of rasGAP¹²⁰ comprising the SH3 domain having amino acidsequence [SEQ ID NO: 6] (NCBI accession number: P20936):

-   -   VRAILPY TKVPDTDEIS FLKGDMFIVH NELEDGWMWV TNLRTDEQGL IVEDLVEEVG        REED

Conversely, it is contemplated that an analogue of the NTF2-like domainof G3BP-2 could be engineered which enables formation of a complexbetween the analogue and an endogenous native target polypeptide ofG3BP-2, thereby competing with G3BP-2 for binding of the endogenousnative target. Suitably, the analogue would also bind an endogenoustarget polypeptide of G3BP-2.

The aforementioned mimetics may be peptides, polypeptides or otherorganic molecules, preferably small organic molecules, with a desiredbiological activity and half-life.

Mimetics may be identified by way of screening libraries of moleculessuch as synthetic chemical libraries, including combinatorial libraries,by methods such as described in Nestler & Liu, 1998, Comb. Chem. HighThroughput Screen. 1 113 and Kirkpatrick et al., 1999, Comb. Chem. HighThroughput Screen 2 211.

It is also contemplated that libraries of naturally-occurring moleculesmay be screened by methodology such as reviewed in Kolb, 1998, Prog.Drug. Res. 51 185.

More rational approaches to designing mimetics may employ computerassisted screening of structural databases, computer-assisted modelling,or more traditional biophysical techniques which detect molecularbinding interactions, as are well known in the art.

Computer-assisted structural database searching is becoming increasinglyutilized as a procedure for identifying mimetics. Database searchingmethods which, in principle, may be suitable for identifying mimetics,may be found in International Purification WO 97/418232 (directed toproducing HIV antigen mimetics), U.S. Pat. No. 5,752,019 andInternational Publication WO 97/41526 (directed to identifying EPOmimetics), each of which is incorporated herein by reference.

Other methods include a variety of biophysical techniques, whichidentify molecular interactions. These allow for the screening ofcandidate molecules according to whether said candidate molecule affectsformation of G3BP-2:endogenous target polypeptide complexes, forexample. Methods applicable to potentially useful techniques such ascompetitive radioligand binding assays (see Upton et al., 1999, suprafor relevant methods), analytical ultracentrifugation, microcalorimetry,surface plasmon resonance and optical biosensor-based methods areprovided in Chapter 20 of CURRENT PROTOCOLS IN PROTEIN SCIENCE Eds.Coligan et al., (John Wiley & Sons, 1997) which is incorporated hereinby reference.

Pharmaceutical Compositions

A further feature of the invention is use of the polypeptide, fragment,variant or derivative thereof as actives in a pharmaceuticalcomposition. The actives may be “immunogenic agents” which are capableof eliciting an immune response in an animal. An immunogenic agent maycomprise a protein, nucleic acid, vaccine or antigen presenting cellloaded or pulsed with an antigen, or any combination of these agents.The antigen presenting cell may be loaded or pulsed with antigen bycontacting the cell with an antigen, for example a protein, polypeptide,fragment, variant or derivative of the invention. The antigen may beinternalised within the antigen presenting cell by any suitable meansincluding for example, phagocytosis, micro-injection, engulfing, and thelike. The antigen may be combined with any suitable carrier, for examplea latex bead, fusion protein, or any other delivery particle commonlyused in the art. The antigen presenting cell may be, for example adendritic cell or any other antigen presenting cell as known in the artof immunology.

A pharmaceutical composition may also comprise an antagonist, whichprevents binding between the NTF2-like domain of G3BP-2 and anendogenous binding partner thereof.

Suitably, the pharmaceutical composition comprises apharmaceutically-acceptable carrier. By “pharmaceutically-acceptablecarrier, diluent or excipient” is meant a solid or liquid filler,diluent or encapsulating substance that may be safely used in systemicadministration. Depending upon the particular route of administration, avariety of carriers, well known in the art may be used. These carriersmay be selected from a group including sugars, starches, cellulose andits derivatives, malt, gelatine, talc, calcium sulfate, vegetable oils,synthetic oils, polyols, alginic acid, phosphate buffered solutions,emulsifiers, isotonic saline, and pyrogen-free water.

Any suitable route of administration may be employed for providing apatient with the pharmaceutical composition of the invention. Forexample, oral, rectal, parenteral, sublingual, buccal, intravenous,intra-articular, intramuscular, intra-dermal, subcutaneous,inhalational, intraocular, intraperitoneal, intracerebroventricular,transdermal and the like may be employed. Intra-muscular andsubcutaneous injection is appropriate for administration of immunogenicagents of the present invention.

Dosage forms include tablets, dispersions, suspensions, injections,solutions, syrups, troches, capsules, suppositories, aerosols,transdermal patches and the like. These dosage forms may also includeinjecting or implanting controlled releasing devices designedspecifically for this purpose or other forms of implants modified to actadditionally in this fashion. Controlled release of the therapeuticagent may be effected by coating the same, for example, with hydrophobicpolymers including acrylic resins, waxes, higher aliphatic alcohols,polylactic and polyglycolic acids and certain cellulose derivatives suchas hydroxypropylmethyl cellulose. In addition, the controlled releasemay be effected by using other polymer matrices, liposomes and/ormicrospheres.

Pharmaceutical compositions of the present invention suitable foradministration may be presented as discrete units such as vials,capsules, sachets or tablets each containing a pre-determined amount ofone or more immunogenic agent of the invention, as a powder or granulesor as a solution or a suspension in an aqueous liquid, a non-aqueousliquid, an oil-in-water emulsion or a water-in-oil liquid emulsion. Suchcompositions may be prepared by any of the methods of pharmacy but allmethods include the step of bringing into association one or moreimmunogenic agents as described above with the carrier, whichconstitutes one or more necessary ingredients. In general, thecompositions are prepared by uniformly and intimately admixing theagents of the invention with liquid carriers or finely divided solidcarriers or both, and then, if necessary, shaping the product into thedesired presentation.

Vaccines

The above compositions may be used as a therapeutic or prophylacticvaccines comprising a polypeptide and/or nucleic acid of the invention,or respective fragments thereof. In one embodiment, the vaccinecomprises an immunogenic agent as described above. Preferably, thevaccine prevents or treats breast cancer.

Accordingly, the invention extends to the production of vaccinescomprising as actives one or more of the immunogenic agents of theinvention. Any suitable procedure is contemplated for producing suchvaccines. Exemplary procedures include, for example, those described inNEW GENERATION VACCINES (1997, Levine et al., Marcel Dekker, Inc. NewYork, Basel Hong Kong) which is incorporated herein by reference.

An immunogenic agent according to the invention can be mixed, conjugatedor fused with other antigens, including B or T cell epitopes of otherantigens. In addition, it can be conjugated to a carrier as describedbelow.

When a haptenic peptide of the invention is used (i.e., a peptide whichreacts with cognate antibodies, but cannot itself elicit an immuneresponse), it can be conjugated with an immunogenic carrier. Usefulcarriers are well known in the art and include for example:thyroglobulin; albumins such as human serum albumin; toxins, toxoids orany mutant cross reactive material (CRM) of the toxin from tetanus,diptheria, pertussis, Pseudomonas, E. coli, Staphylococcus, andStreptococcus; polyamino acids such as poly(lysine:glutamic acid);influenza; Rotavirus VP6, Parvovirus VP1 and VP2; hepatitis B virus coreprotein; hepatitis B virus recombinant vaccine and the like.Alternatively, a fragment or epitope of a carrier protein or otherimmunogenic polypeptide may be used. For example, a haptenic peptide ofthe invention can be coupled to a T cell epitope of a bacterial toxin,toxoid or CRM. In this regard, reference may be made to U.S. Pat. No.5,785,973 which is incorporated herein by reference.

The vaccines can also contain a physiologically-acceptable carrier,diluent or excipient such as water, phosphate buffered saline andsaline.

The vaccines and immunogenic agents may include an adjuvant as is wellknown in the art. Suitable adjuvants include, but are not limited toadjuvants for use in human for example SBAS2, SBAS4, QS21 or ISCOMs.

The immunogenic agents of the invention may be expressed by attenuatedviral hosts. By “attenuated viral hosts” is meant viral vectors that areeither naturally, or have been rendered, substantially avirulent. Avirus may be rendered substantially avirulent by any suitable physical(e.g., heat treatment) or chemical means (e.g., formaldehyde treatment).By “substantially avirulent” is meant a virus whose infectivity has beendestroyed. Ideally, the infectivity of the virus is destroyed withoutaffecting the polypeptides that carry the immunogenicity of the virus.From the foregoing, it will be appreciated that attenuated viral hostsmay comprise live viruses or inactivated viruses.

Attenuated viral hosts which may be useful in a vaccine according to theinvention may comprise viral vectors inclusive of adenovirus,cytomegalovirus and preferably pox viruses such as vaccinia (see forexample Paoletti and Panicali, U.S. Pat. No. 4,603,112 which isincorporated herein by reference) and attenuated Salmonella strains (seefor example Stocker, U.S. Pat. No. 4,550,081 which is hereinincorporated by reference). Live vaccines are particularly advantageousbecause they lead to a prolonged stimulus that can confer substantiallylong-lasting immunity.

Multivalent vaccines can be prepared from one or more different epitopesof G3BP-2.

A recombinant vaccinia virus may be prepared to express a nucleic acidaccording to the invention. Upon introduction into a host, therecombinant vaccinia virus expresses the immunogenic agent, and therebyelicits a host CTL response. For example, reference may be made to U.S.Pat. No. 4,722,848, incorporated herein by reference, which describesvaccinia vectors and methods useful in immunization protocols.

A wide variety of other vectors useful for therapeutic administration orimmunization with the immunogenic agents of the invention will beapparent to those skilled in the art from the present disclosure.

The nucleic acid of the invention may be used as a vaccine in the formof a “naked DNA” vaccine as is known in the art. For example, anexpression vector of the invention may be introduced into a mammal,where it causes production of a polypeptide in vivo, against which thehost mounts an immune response as for example described in Barry, M. etal., (1995, Nature, 377:632-635) which is hereby incorporated herein byreference.

Dendritic Cell Therapy

“Dendritic cells” (DC) are antigen presenting cells capable ofinitiating an antigen-specific T-cell response in an animal. DC's may beisolated from various locations of an animal's body, includingperipheral blood. Methods for in vitro proliferation and expansion of DCprecursors have been described, for example in U.S. Pat. No. 5,994,126,incorporated herein by reference. Also described is a method forproducing mature dendritic cells in culture from proliferating dendriticcell precursors.

“Dendritic cell therapy” refers to therapeutic cancer vaccines orcellular vaccines used for tumour immunotherapy as a method for treatingcancer. Dendritic cell therapy typically involves isolating DC from apatient, culturing the isolated DC in the presence of atumour-associated antigen (TAA) thereby contacting the DC with a TAA(“antigen loading or pulsing”), and administering the antigen loadedDC's to the patient. Other methods for antigen loading an isolated DCinclude transfecting, micro-injecting, calcium phosphate transfection,DEAE-transfection, electroporation or otherwise introducing an isolatednucleic acid encoding a tumour-associated antigen into the isolated DC.Common method for introducing nucleic acids into a cell are described inChapter 9 of CURRENT PROTOCOLS IN MOLECULAR BIOLOGY Eds. Ausubel et al.,(John Wiley & Sons, Inc. 1995-1999), incorporated herein by reference.Preferably, the TAA is G3BP-2, fragment, variant, homolog or derivativethereof. The nucleic acid may be DNA or RNA. The nucleic acid may betransiently or stably express the TAA as is known in the art.

Methods for loading or pulsing dendritic cells with an antigen aredescribed in (Meidenbauer et al, 2001, Biol Chem 4 507; Rains, et al,2001, Hepatogastroenterology 38 347; Nestle, 2000, Oncogene 56 6673;Gilboa and Lyerly, 1998, Cancer Immunotherapy 46 82) incorporated hereinby reference.

U.S. Pat. No. 5,788,963, incorporated herein by reference, describesmethods and compositions for use of human dendritic cells to activateT-cells for immunotherapeutic responses against primary and metastaticprostate cancer. In one embodiment isolated DC are exposed in vitro to aprostate cancer antigen before administration to a patient.

In one embodiment of the present invention, a pharmaceutical compositioncomprises DC's antigen load with G3BP-2 polypeptide, fragment, variantor derivative thereof in accordance with the invention. In anotherembodiment of the present, the DC's are transfected with a nucleic acidencoding a G3BP-2 polypeptide, fragment, variant or derivative thereof.Suitable G3BP-2 protein fragments for use with DC cell therapy are setforth as SEQ ID NOS: 1 and 2. It will be appreciated by a skilled personthat antigen presenting cells other than DC may be used and that use ofDC is merely preferred.

Preparation of Immunoreactive Fragments

The invention also extends to a method of identifying an immunoreactivefragment of a polypeptide, variant or derivatives according to theinvention. This method essentially comprises generating a fragment ofthe polypeptide, variant or derivative, administering the fragment to amammal; and detecting an immune response in the mammal. Such responsemay include production of elements which specifically bind G3BP-2,respective variant, or derivative thereof, including NTF2-like domain,which may provide a protective effect against breast cancer.

Prior to testing a particular fragment for immunoreactivity in the abovemethod, a variety of predictive methods may be used to deduce whether aparticular fragment can be used to obtain an antibody that cross-reactswith the native antigen. These predictive methods may be based onamino-terminal or carboxy-terminal sequence as for example described inChapter 11.14 of Ausubel et al., supra. Alternatively, or in addition,these predictive methods may be based on predictions of hydrophilicityas for example described by Kyte & Doolittle 1982, J. Mol. Biol. 157 105and Hopp & Woods, 1983, Mol. Immunol. 20 483) which are incorporated byreference herein, or predictions of secondary structure as for exampledescribed by Choo & Fasman, 1978, Ann. Rev. Biochem. 47 251), which isincorporated herein by reference.

Generally, a peptide fragment consisting of 10 to 15 residues providesoptimal results. Peptides as small as 6 or as large as 20 residues haveworked successfully. Such peptide fragments may then be chemicallycoupled to a carrier molecule such as keyhole limpet hemocyanin (KLH) orbovine serum albumin (BSA) as for example described in Sections 11.14and 11.15 of Ausubel et al., supra).

The peptides may be used to immunize an animal as for example discussedabove. Antibody titers against the native or parent polypeptide fromwhich the peptide was selected may then be determined by, for example,radioimmunoassay or ELISA as for instance described in Sections 11.16and 11.14 of Ausubel et al., supra.

Immunoreactive protein fragments in the context of the MajorHistocompatibility Complex (MHC) may be determined using methods wellknown in the art including those described by Schultze and Vonderheide,2001, incorporated herein by reference.

Detection Kits

The present invention also provides a kit for detection of G3BP-2 in abiological sample. A kit will contain one or more particular agentsdescribed above depending upon the nature of the test method employed.In this regard, the kits may include one or more of a polypeptide,fragment, variant, derivative, antibody, antibody fragment or nucleicacid according to the invention. The kits may also optionally includeappropriate reagents for detection of labels, positive and negativecontrols, washing solutions, dilution buffers and the like. For example,an antibody-based detection kit may include (i) a polypeptide, orfragment or variant thereof according to the invention (which may beused as a positive control), (ii) an antibody according to the invention(preferably a monoclonal antibody) which binds to G3BP-2 or fragmentthereof in (i), and (iii) a suitable means for detecting a complexformed between a target (eg. G3BP-2 in a sample) and the antibody in(ii), the detection means may include, for example colloidal gold.Suitable antibodies for use in a detection kit include those describedherein in relation to Western blots and immunohistochemistry.

A detection kit may also be nucleic acid based. Such a detection kit mayinclude the step of amplifying a nucleic acid from the test sampleobtained from an animal using techniques such as Polymerase ChainReaction (PCR) or other known amplification method known in the art.Useful PCR primers may include those set forth herein as SEQ ID NOS:16-21. The nucleic acid may be RNA or DNA. The test sample is preferablybreast tissue isolated from a patient.

Throughout this specification, unless the context requires otherwise,the words “comprise”, “comprises” and “comprising” will be understood toimply the inclusion of a stated integer or group of integers but not theexclusion of any other integer or group of integers.

In order that the invention may be readily understood and put intopractical effect, particular preferred embodiments will now be describedby way of the following non-limiting examples.

EXAMPLE 1

Cloning, Sequence Homology and Structural Homology

PCR and Subcloning

PCR reactions to amplify probes or for hybrid mapping were carried outusing 1.1 units of Tth Plus DNA Polymerase fragment (BiotechInternational) and buffer supplied by the manufacturer (BiotechInternational) and contained 100 ng template DNA and 50 pmol of eachappropriate primer. Cycling conditions were: denaturation of DNA at 94°C. for 1 min, annealing at 65° C. for 1 min and extension at 72° C. for1 min for 25 cycles. Primers that can be used to amplify either fulllength G3BPs or the NTF2-like domain of the G3BPs:

Full length human G3BP-1 is amplified using primers G3BP-2met withG3BP-1stop; full length human G3BP-2 is amplified using primersG3BP-2met with G3BP-2stop; the NTF2-like domain of human G3BP-1 isamplified using primers G3BP-1 met with G3BP-1 ntf; and the NTF2-likedomain of human G3BP-2 is amplified using primers G3BP-2met withG3BP-2ntf.

Primer Sequences: G3BP-1met atggtgatggagaagcctagtcccctgctggt [SEQ IDNO:16] G3BP-2met atggttatggagaagcccagtccg [SEQ ID NO:17] G3BP-2metatcaagttcaggctcagaatcacc [SEQ ID NO:18] G3BP-1metctgaggctcagtgacaaacccaac [SEQ ID NO:19] G3BP-2stopgcttcagcgacgctgtcctgtgaagc [SEQ ID NO:20] G3BP-1stopctgccgtggcgcaagcccccttcc [SEQ ID NO:21]Library Screening and Phage Isolation

Plasmid preparations, to be used as probes, library screening orsequencing, were made as described by Sambrook et al., 1989 (Sambrook etal., 1989). The libraries, late-primitive-streak stage mouse embryo cDNA(Kennedy et al. 1997) and a foetal human brain (cDNA kindly donated byDr. T. Cox, University of Adelaide, Australia) were plated out atapproximately 25 000 pfu/130 mm plate and duplicate filters (Hybond-N,Amersham) made from 21 plates. Library screening was performed asdescribed by Sambrook et al., 1989 (Sambrook et al. 1989) withradioactive probes prepared using Amersham's Megaprime DNA labelingsystem according to the manufacturer's instructions. Hybridizationconditions were as follows: 50% formamide, 5×SSC, 20 mM Tris.HCl pH 7.6,1× Denhardt's solution and 0.1% SDS at 42° C. A minimum of 3×20 minwashes of hybridized filters were performed in 0.2×SSC, 0.1% SDS at 65°C. cDNAs from purified plaques were subcloned into pBluescript SK(Stratagene).

EXAMPLE 2

Protein Expression in Insect Cells

Sub Cloning and Expression of Proteins Using Baculovirus

Full length cDNAs encoding for MmuG3BP-2a and 2b were excised frompBluescript using EagI (−85 bp from met start codon) and AccI (+140 bpfrom the stop codon) and subsequently end filled using Klenow fragmentpolymerase. These cDNAs were subcloned into SmaI linearised pBacPAK9(Clontech) and transformed into competent DH5′α E. coli cells. Theorientation of the inserts were checked by PCR. Plasmids containing theinserts in the correct orientation were transfected into Spodopterafrugiperda IPLB-Sf21 (Sf21) cells with Bsu36I digested BacPAK6 viralexpression vector according to the manufacturers instructions (Clontech#K1601-1). Recombinant virus plaques were selected from an Sf21monolayer and once again screened by PCR. Virus containing the codingcDNAs were amplified in Sf21 cells and total cell lysates visualised onpolyacrylamide gels for expression of proteins.

EXAMPLE 3

Mapping of G3BP-rasGAP¹²⁰ Interactions

Fusion Protein Constructs and Expression

Truncated cDNAs representing either N-terminal or C-Terminal sequencesof G3BP-1 and G3BP-2a (FIG. 2) were subcloned into bacterial GST-fusionexpression vectors (pGex, Pharmacia) so that the recombinant fusionproteins could be expressed and used in bead binding assays (see below)to identify the sub-domains of G3BP that interact with the SH3 domain ofrasGAP¹²⁰. The vectors containing the recombinant fusion proteinconstruct were transformed into competent DH5′ E. Coli and expressed byIPTG induction. To collect the recombinant proteins the cells werewashed and resuspended in PBS and sonicated to release the fusionproteins.

The N-terminal domain of rasGAP¹²⁰ (kindly provided by Prof. I.G.Macara, University of Virginia) containing amino acids 1-356 whichincludes the full SH3 domain of rasGAP¹²⁰ was subcloned into proEX HTbacterial expression vector (Life Technologies) and expressed asdescribed above.

Bead Binding Assays

Glutathione columns (Pharmacia) were blocked overnight at 4° C. in 1×binding buffer A (50 mM HEPES pH 7.4, 100 mM NaCl, 5 mM MgCl₂, 50 mg/mlBSA, 1 mM DTT, 1 mM PMFS and protease inhibitors (Protease inhibitorcocktail, Roche Diagnostics, Australia). The following day the columnswere washed twice with binding buffer A. Bacterial lysates containingeither expressed GST alone or GST fusion proteins were diluted 1:1 with2× binding buffer A and added to the equilibrated GST columns, thecolumns were gently rocked overnight at 4° C. To remove excess proteinsthe columns were washed twice in 1× binding buffer A. Bacterial lysatescontaining the His-tagged N-terminal of rasGAP¹²⁰ protein were combinedwith an equal volume of 2× binding buffer A containing 200 mg/ml BSA and0.1% Tween. In addition to the high concentration of BSA, which is usedto block non-specific protein-protein interactions, Ethidium bromide wasadded to a final concentration of 25 ng/ml to abrogate non-specificinteractions caused by excess bacterial genomic DNA and bacterial RNA.This mix was then added to the pre-bound GST columns (above) and allowedto bind for 2 hrs at 4° C. The columns were then washed three times withbinding buffer B (50 mM HEPES pH 7.4, 200 mM NaCl, 5 mM MgCl₂, 1 mM DTT,1 mM PMFS and protease inhibitors).

Western Analysis of Protein-Protein Interactions

One μl of the GST beads complexed with proteins, as described above, wasadded to an equal volume of 2×PAGE loading dye, heated for 5 mins at 95°C. and loaded onto a 7.5% Polyacrylamide gel and run at 100V for 3hours. The proteins were transferred to Nitrocellulose and probed withanti-His antibodies (Tetra-His antibody, Qiagen). Columns, which werebound with GST alone, were used as negative controls to ensure nonon-specific His-tagged N-terminal rasGAP¹²⁰ remained associated withthe columns. To ensure that the GST columns had been saturated with theappropriate GST-G3BP peptides all experiments were probed with anti-GSTto confirm that the columns contained the “bait” peptide (data notshown).

Protein Purification

500 ml of Sf21 cells at a concentration of 1.2 to 2.0×10⁶ cells/ml cellswere infected with 60 ml of baculovirus (6.6×10⁸ to 1.6×10⁹ pfu/ml)containing cDNAs for expression of proteins and incubated at 28° C. for4 days in an orbital incubator. The cells were harvested and washedtwice at 4° C. with PBS, lysed in 50 ml of HNTG lysis buffer by 30 secvortexing and gentle rocking for 30 min at 4° C. and cleared bycentrifugation at 9500 rpm×10 min at 4° C. The final salt concentrationwas adjusted to 30 mM NaCl by addition of NaCl free and triton X-100free HNTG lysis buffer (containing protease inhibitors) and incubatedovernight on a rotating mixer at 4° C. with pre-equilibrated heparinsepharose CL-6B (Pharmacia biotech #17-0467-01) at a concentration of 15mg of protein/ml of heparin sepharose. The gel was washed in 50 mM HepespH 7.5, 10% glycerol and packed into a column (Pharmacia XK-26). Thecolumn was subjected to a 30 mM to 1.0 M NaCl gradient over 120 min at aflow rate of 0.83 ml/min using a Pharmacia FPLC system. 1.5 ml sampleswere collected and assayed for MmuG3BP by separation on polyacrylamidegels and visualised by coomassie blue staining or Western blot analysisusing the 663 antibody.

Fractions containing the recombinant G3BP proteins were pooled and againdiluted to a final 60 mM NaCl concentration. The pooled samples wereincubated with agarose-polyribouridylic acid AGPoly(U), type 6(Pharmacia biotech #27-5535) at a concentration of 1.5 mg of protein/mlof gel overnight at 4° C. on a rotating mixer. The following morning thegel was washed with 50 mM Hepes pH 7.5, 10% glycerol, packed into aglass column (Pharmacia XK-16) and subjected to a 60 mM to 1 M NaClgradient at a flow rate of 0.33 ml/min as described above. 0.5 mlfractions were collected, assayed and pooled as described above. Pooledsamples were diluted to 30 mM NaCl as described above and loaded onto amono S HR 5/5 ion exchanger column (Pharmacia #17-0547-01) at a flowrate of 0.3 ml/min and subjected to a 30 mM to 1 M NaCl gradient. 0.5 mlfractions were collected and assayed as described above.

EXAMPLE 4

G3BP-2 Anti-Sera

Polyclonal Antibody Production

Affinity purified antibodies to G3BP-2 were obtained from an antiserumraised against an internal peptide sequence (SATPPPAEPASLPQEPPKPRV).Polyclonal antibodies raised against G3BP-1 have been describedelsewhere (Parker et al. 1996) and a monoclonal G3BP-1 antibody iscommercially available as (BD Biosciences, Sydney, AUS).

G3BP-2 Antibody Specificity

The specificity of the G3BP-2 antibody was assessed by testing itsability to bind human recombinant GST fusion G3BP-1, G3BP-2a, G3BP-2band G3BP-2a truncations. LB/Amp agar plates were inoculated with E. colitransformed with pGEX vectors (Amersham Biosciences, GST gene fusionsystem) containing four alternative truncations of G3BP-2a (N1, N2, C1and C2), as well as full length G3BP-1, 2a and 2b, as previouslydescribed in Kennedy et al. (2001). One colony from each plate was usedto inoculate 5 ml of LB/Amp broth, which was incubated at 37° C.overnight. Isolation of the GST fusion proteins was performed byglutathione sepharose affinity chromatography from IPTG-induced culturesas per the manufacturer's instructions (Amersham Biosciences). Followingelution with glutathione, the solutions were spun at 5000 rpm for 5minutes and the supernatant was dialysed in PBS.

Purified recombinant G3BP-1, G3BP-2a, G3BP-2b, and the four truncationsof G3BP-2a were resolved by 12% SDS-PAGE and transferred to PVDF(Millipore) and incubated with anti-G3BP-2 antibody. Proteins werevisualised by HRP conjugated anti-rabbit antibodies using an ECL system(Amersham Biosciences).

Cell Extracts, SDS-PAGE and Western Blotting

The expression of G3BP in human cell lines was examined by Western blot.Cells were maintained in vitro in DMEM supplemented with 10% FCS andharvested by trypsinisation, washed twice with PBS and resuspended inHNTG buffer (50 mM Hepes, pH 7.5, 150 mM NaCl, 1% Triton X-100, 10%glycerol, 1 mM MgCl₂, 1 mM EGTA, 1 mM Na₃VO₄, 10 mM Na₄P₂O₇, 10 mM NaF,1 mM PMSF and 1× mammalian protease inhibitor cocktail # P8340 (Sigma,Castle Hill, AUS). Lysates were cleared by centrifugation at 15 000 rpmfor 10 minutes and the protein concentration was determined using thePierce BCA Protein Assay (Rockford, USA). A total of 75 μg of proteinwas resolved by 8% SDS-PAGE and transferred to an Immobilon-P PVDFmembrane (Millipore, Sydney, AUS) for Western analysis using theantibodies described above. Proteins were visualised by horseradishperoxidase (HRP) conjugated anti-rabbit or mouse antibodies using an ECLsystem (Amersham Biosciences, Sydney, AUS).

Antibodies Used for Immunohistochemistry were Highly Specific

To assess antibody specificity and the expression of G3BPs in a range ofcell types, the breast cancer cell line, MDA-MB-435 and the cervicalcancer cell line, HeLa were lysed and equal amounts of protein wereresolved by SDS-PAGE. The samples were transferred to a membrane andanalysed by Western blotting. The commercial monoclonal G3BP-1 antibody(BD Biosciences) was used to assess G3BP-1 expression, while thepolyclonal G3BP-2 antibody (Kennedy et al. 2001) was used to examine theexpression of G3BP-2 in these cell lines. As illustrated in FIG. 9,Panel A, it is apparent that both cell lines express significant levelsof G3BP-1, present as a single distinct band, and G3BP-2, present as twodistinct bands, representing the two different isoforms. The sameexpression patterns were seen in the immortalised human cell line HEK293T (data not shown). There did not appear to be any significantcross-reactivity. However, there was some variation between the relativemasses of G3BP-1, 2a and 2b according to amino acid sequence, and theapparent masses of the proteins as determined by PAGE. According to thedata, G3BP-2a should resolve at a point just above G3BP-1, but itactually appears just below G3BP-1.

The specificity of the polyclonal G3BP-2 antibody was further examined.It was tested against recombinant GST-fusion G3BP-1, 2a and 2b as wellas several different truncated forms of G3BP-2. As presented in FIG. 9,Panel B, the anti-G3BP-2 antibody specifically binds to recombinantG3BP-2a and G3BP-2b (lanes 3 and 1, respectively) while it does not bindto G3BP-1 (lane 2). The antibody bound two of the recombinant G3BP-2atruncations (lanes 5 and 7) but it did not bind the short C-terminal orN-terminal truncations (Lanes 4 and 6). This indicated that the regionthat the antibody binds to is the central domain as shown in FIG. 9,Panel C. It was found that the G3BP-2 antibody is specific for G3BP-2aand 2b and binds the central region of the protein only. Despiteexcessively high protein loads (3 μg of recombinant protein per lane),the antibody did not cross-react with G3BP-1 or the shorter of theG3BP-2 N-terminal (N1) or C-terminal (C2) truncations.

EXAMPLE 5

G3BPs have a Tissue Specific Expression

Western Blotting

Proteins were fractionated by sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis (7.5% gel) using the method ofLaemmli and transferred onto polyvinylidene difloride (PVDF) membrane(Millipore Corp.) in a Bio-Rad Trans-blot Cell using a transfer buffercontaining 25 mM Tris pH 8.3, 192 mM Glycine and 15% methanol.Electroblotting was carried out at 100 volts overnight at 4° C. The blotwas blocked by incubation in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1%Tween-20 containing 10% skim milk (blocking solution) at roomtemperature for 1 hour, followed by incubation with the primaryantibody, anti-G3BP-1 (diluted 1:500) or anti-G3BP-2 (diluted 1:4000) inblocking solution, at 4° C. overnight. The blot was washed 3 times inblocking solution for 10 minutes and subsequently incubated for 2 hoursat 37° C. in the secondary antibody which was an anti-rabbit conjugatedwith horseradish peroxidase (BioRad), diluted 1:10000 in blockingsolution.

Total Protein Cell and Tissue Lysates

Cells and tissues were washed twice with ice cold phosphate-bufferedsaline (PBS) and solubilised or homogenised with a dounce homogeniser inHNTG lysis buffer consisting of 50 mM HEPES, pH 7.5, 150 mM NaCl, 1%Triton X-100, 10% glycerol, 1 mM MgCl₂, 1 mM EGTA, phosphataseinhibitors (1 mM Na₃VO₄, 10 mM Na₄P₂O₇ and 10 mM NaF) and proteaseinhibitors (1 μg of leupeptin per ml, 1 μg of trypsin inhibitor per ml,1 μg of pepstatin A, 2 μg of aprotinin per ml, 10 μg benzamidine per ml,1 mM phenylmethylsulfonyl floride, 1 μg of antipain per ml, 1 μg ofchymostatin per ml). Lysates were cleared by centrifugation at 15 000rpm for 10 min and the protein concentration determined by the Bradforddye-binding procedure using Bio-Rads Protein Assay (# 500-0001).

Immunohistochemistry

Immunohistochemistry was used to analyse the degree of cell specificityof the G3BP-1 and G3BP-2 expression. Until isoform specific antibodiesare raised against G3BP-2a and G3BP-2b it is not possible to distinguishthese isoforms in immunohistochemistry, however, in some tissues a itcan be determined which specific isoform is being expressed bycomparison to the Western blot data (as herein described).

FIGS. 6A and 6B show a cross section of results from some of the tissuesstudied. Panels A to E are probed with anti-G3BP-1 antibodies whereaspanels F to J are probed with anti-G3BP-2 antibodies. Panels A and Fshow a comparison of adult mouse brain. As determined by Westernanalysis (FIG. 5, panel A), brain does not express G3BP-1 (FIG. 6, panelA) however, a sub-population of cells express G3BP-2a (panel F).

The inventors determined by cell morphology and double staining with aneural marker (data not shown) that the G3BP-2a positive cells areneural cells (panel F, labeled Ne) and that the negative cells are glialcells (panel F, labeled GI). In the kidney (panels B and C) G3BP-1appears to be expressed in interstitial cells or a sub-population oftubules (panel B) whereas G3BP-2 is expressed at low levels in alltubules (labeled Tu in panel G). Neither G3BP-1 nor G3BP-2 are expressedin the glomerulus (labeled Gm in panel G). The colon (panels C and H)shows that G3BP-1 is expressed at the periphery of the intestinal glandsor possibly in interstitial cells whereas G3BP-2 is expressed in thelumen of the intestinal glands (labeled Ig in panels H and I). G3BP-2 isalso expressed at high levels in the villi of the small intestine (FIG.6 panel I) whereas G3BP-1 (FIG. 6 panel D) was not detected at levelsabove the background staining of the negative control. Once again nodetectable staining was observed for G3BP-1 in stomach (FIG. 6 panel E)whereas G3BP-2 (presumably G3BP-2b only from the Western blot data)appears to be expressed in the mucus secreting cells of the stomachlumen (labeled Ep in panel J) and the internal surface of the pyloricglands (labeled Pg in panel J). Other tissues examined byimmunohistochemistry include heart, liver and spleen (data not shown).Heart and liver showed a general low level expression of G3BP-1 andG3BP-2 whereas spleen was negative for G3BP-2 and showed a cell specificstaining of G3BP-1. It is still to be determined what types of cellsconstitute the G3BP-1 expressing islands observed within the spleen.

Frozen mouse tissue sections (10 μm thickness) were fixed to Histogriptreated slides (SuperFrost Plus microscope slides, Menzel-Glaser,Germany) and air-dried overnight at room temperature. Sections werefixed for 5 minutes in 50% Chloroform, 50% Acetone, air dried andrehydrated in Tris-buffered saline (TBS) (25 mM Tris, 137 mM NaCl, pH7.4). Nonspecific antibody binding was inhibited by incubation with TBScontaining 4% skim milk powder for 15 minutes followed by an additional20 min incubation in TBS containing 10% normal goat serum (Gibco).Sections were then incubated overnight with either anti-G3BP-1 (diluted1:300) or an anti-G3BP-2 (diluted 1:2000). Excess antibody was removedby washing in TBS (3×5 min) and prediluted horseradish peroxidase (HRP)labeled anti-rabbit immunoglobulins (Envision) was applied for 30minutes. Sections were then washed with TBS (3×5 min) and colour wasdeveloped in 3,3′-diaminobenzidine with H₂O₂ (Zymed) as a substrate for2 minutes. Sections were washed with gently running tap water for 10minutes to remove excess chromogen, lightly counterstained in Mayers'haematoxylin, dehydrated through ascending graded alcohols, cleared inxylene, then mounted using DPX (Harlow and Lane 1988).

EXAMPLE 6

Chromosomal Location of G3BPs

Fluorescence In Situ Hybridisation of PACs

Fluorescence in situ hybridisation (FISH) was performed on peripheralhuman metaphase chromosomes. PAC DNA was biotin-14dATP-labelled by nicktranslation using the BioNick labeling system (Life Technologies).Chromosome preparation and FISH conditions were as described previously(Wicking et al., 1995). Slides were analysed using an Olympus BH2fluorescent microscope.

Chromosomal Mapping

Two HsaG3BP-1 specific primers, 5′ GGAGGCATGGTGCAGAAACCA [SEQ ID NO: 12]and 5′ CAGGAAAGGGAAGAGAGGGAG [SEQ ID NO: 13] and two HsaG3BP-2 specificprimers, 5′ GTCTTGGCAGTGGTACATTAT [SEQ ID NO: 14] and 5′AGTTCACTTTGTCGTAGATAGTTTAAG [SEQ ID NO: 15] were used to amplifyspecific templates from human genomic DNA and were subsequently used onthe Genebridge4 Hybrid panel to identify positives. This data was thenprocessed by the online mapping software available through the WhiteheadInstitute/MIT Center for Genome research (http://carbon.wi.mit.edu).

EXAMPLE 7

Antagonists of G3BP-2

An antagonist which prevents or disrupts G3BP-2 binding with itsendogenous target may be useful in preventing or treating breast cancer.An antagonist may mimic either the NTF2-like domain of G3BP-2 or mimican endogenous target which binds to the NTF2-like domain. For example,the SH3 domain of rasGAP¹²⁰ could be used as a peptide antagonist forblocking the activity of G3BP-2 in breast cancers. The amino acidsequence of the SH3 domain of rasGAP¹²⁰ is: VRAILPY TKVPDTDEISFLKGDMFIVH [SEQ ID NO:6] NELEDGWMWV TNLRTDEQGL IVEDLVEEVG REEDThe NCBI accession number: P20936 for the above sequence. Theantagonists may be a polypeptide, but may also be a non-peptide moleculewhich is capable of acting as an antagonist.

Mimetics may be identified by way of screening libraries of moleculessuch as synthetic chemical libraries, including combinatorial libraries,by methods such as described in Nestler & Liu, 1998, supra andKirkpatrick et al., 1999, supra. Libraries of naturally-occurringmolecules may be screened by methodology such as reviewed in Kolb, 1998,supra.

Three-dimensional (3D) structural modelling by homology can be used toassign a 3D structure to the NTF2-like domain of G3BP-2 based onstructural information from the known crystal structure of the NTF-2polypeptide. Methods for 3D structural modelling by homology isdescribed in Blundell et al, 1987, Nature 326 347, herein incorporatedby reference. An antagonist that interacts with the NTF2-like domain maybe designed based on structural modelling by homology.

Mimetics may be designed using computer assisted screening of structuraldatabases, computer-assisted modelling, or more traditional biophysicaltechniques which detect molecular binding interactions, as are wellknown in the art.

Other methods include a variety of biophysical techniques which identifymolecular interactions. These methods may screen candidate moleculesaccording to whether the candidate molecule affects formation ofG3BP-2:endogenous target polypeptide complexes. Methods applicable topotentially useful techniques such as competitive radioligand bindingassays (see Upton et al., 1999, supra for relevant methods), analyticalultracentrifugation, microcalorimetry, surface plasmon resonance andoptical biosensor-based methods are provided in Chapter 20 of CURRENTPROTOCOLS IN PROTEIN SCIENCE Eds. Coligan et al., (John Wiley & Sons,1997) which is incorporated herein by reference.

EXAMPLE 8

Diagnosis of Breast Cancer

G3BP-2 may be useful for diagnosing breast cancer in an individual. Inone embodiment, the method may include the steps of: (i) assaying a testsample obtained from the mammal for expression of G3BP-2 polypeptide;(ii) comparing G3BP-2 expression from the test sample with expression ina normal sample from a normal mammal; and (iii) diagnosing the mammalwith a likelihood of breast cancer if the expression of G3BP-2 in thetest sample is different than the normal sample. The term differentrefers to at least a detectable difference either by aided or unaidedmeans. For example, an unaided means includes a person visuallycomparing a difference in relative apparent abundance of protein, suchas a thicker or darker “band” on a Western blot or darker well of anELISA. Aided means includes for example, use of a microscope to assessantibody binding of a tissue section or an apparatus that is capable isdetecting and measuring a difference in protein amount, for example anELISA plate reader or FACS. The method for diagnosing breast cancer mayinclude the step of detecting a G3BP-2 polypeptide, or fragment thereof,in the test sample using an antibody which binds to the G3BP-2polypeptide, or fragment thereof. The antibody described herein, forexample as used in FIGS. 7-9, may be useful in a diagnostic kit. Theantibody may be a polyclonal or monoclonal antibody.

The method for diagnosing breast cancer may include methods of detectinga polypeptide, for example Western blot analysis, ELISA, FACS analysisand immunohistochemistry as is commonly known in the art. Examples ofWestern blot analysis and immunohistochemistry as described herein maybe useful in detecting G3BP-2 polypeptide, fragment, homolog orderivative thereof. Western blot analysis is useful in determiningexpression of different isoforms of G3BP-2, ie. G3BP-2a and G3BP-2b,which are distinguishable by size, as shown for example in FIG. 5.Immunohistochemistry is useful in determining cellular and subcellularlocalisation of G3BP-2, as shown for example in FIGS. 6A and 6B. Anantibody which specifically binds to either G3BP-2a or G3BP-2b is usefulin determining expression respective G3BP-2 isoform.

Detection of G3BP-2 Protein in Human Cancer by Immunohistochemistry

Patients

Fifty-nine cases of invasive breast carcinoma diagnosed at theDepartment of Pathology, Royal Brisbane Hospital, between 1981 and 1990were randomly selected. Archival paraffin blocks were accessed subjectto ethics approval from the Royal Brisbane Hospital and had beenpreviously characterised as part of a larger study of MUC1 epithelialmucin expression (McGuckin et al. 1995). Histological classification andgrading of the tumours was performed in accordance with the Nottinghammodification of the Bloom and Richardson system (Elston & Ellis 1990).Data including nodal status and oestrogen receptor (ER) status asdetermined by biochemical dextran-coated charcoal method were obtainedfrom clinical charts and pathology records.

Immunohistochemistry of Breast Cancer Sections

Breast tumour sections (3-4 μm) were affixed to adhesive slides anddried at 37° C. overnight. The sections were dewaxed and rehydratedthrough descending graded alcohols to deionised water using standardprotocols. Sections to be stained for G3BP-1 were subjected to antigenheat retrieval by autoclaving at 120° C. for 20 minutes in 1 mM EDTA, pH8.0. G3BP-2 samples were subjected to antigen heat retrieval by boilingin 0.1M Tris-HCl, pH 9.0-9.2, for 5 minutes in a microwave, andrepeating the process using fresh Tris-HCl buffer. All sections wereallowed to cool to room temperature (20-30 min) and then washed in Trisbuffered saline, pH 7.4 (TBS). Endogenous peroxidase activity wasblocked by incubating the section in 1.0% H2O2, 0.1% NaN₃ in TBS for 10minutes. Sections were washed in TBS and subsequently incubated in 4%non-fat skim milk powder in TBS for 15 minutes. Sections were rinsedbriefly in TBS and then incubated with 10% normal goat serum (NGS) for20 minutes in a humidified chamber. Excess normal serum was decanted andprimary antibody (or TBS as negative control) was applied overnight atroom temperature. Sections were washed in TBS and then incubated withsecondary antibody (DAKO, Glostrup Denmark, EnVision Kit) for 45minutes. Sections were washed in TBS and colour was developed in3,3-diaminobenzidine (DAB) with H₂O₂ as substrate. Sections were washedin gently running tap water then lightly counterstained in Mayers'haemotoxylin, dehydrated through ascending graded alcohols, cleared inxylene, and mounted in DPX mounting medium.

G3BP-2 is Over-Expressed in 88% of Breast Tumours

In addition to determining expression of G3BP-2, expression of G3BP-1was also examined in 24 breast tumour cases by immunohistochemistry asshown in FIG. 8. Of these, 22 sections were infiltrating ductalcarcinomas (IDC) and two were cases of infiltrating lobular carcinoma(ILC). All sections were counterstained with haematoxylin, which stainsnuclei blue and the expression of G3BP-1 was visualised usinghorseradish peroxidase seen as brown staining (See FIG. 8, Panels A-C).Most normal cells exhibited detectable cytoplasmic expression of G3BP-1(see FIG. 8, Panel C). Two normal ducts (ND) are seen in FIG. 8, PanelC, and cytoplasmic expression of G3BP-1 is apparent as seen by thedistinct brown Panel A and B, but the adjacent connective tissue (CT)does not express G3BP-1 at detectable levels. The tumour cells in FIG.8, Panels A and B appear to express higher levels of G3BP-1 in thecytoplasm as compared to that seen in the normal ducts of FIG. 8, PanelC.

In many cases tumour staining was heterogeneous and in some cases G3BP-1appeared to localise more prominently to one side of the cell. This canbe seen quite clearly in some of the tumour cells in FIG. 8, Panel A(indicated by the arrow). There was no nuclear staining present in anynormal cells, although two of the 24 tumour cases contained distinctnuclear staining in less than 10% of tumour cells.

In summary, most normal breast cells expressed G3BP-1, but all of thetumours examined appeared to over-express G3BP-1 to some extent (seeTable 3). No significant relationship was found between G3BP-1over-expression and clinicopathological parameters of breast cancer suchas lymph node involvement, hormone receptor status or nuclear orhistological grade.

A total of 58 breast tumour cases were examined by immunohistochemistryfor altered G3BP-2 expression. Of these, 54 tumours were IDCs and fourwere ILCs. As with G3BP-1, all sections were counterstained withhaematoxylin and the expression of G3BP-2 was visualised usinghorseradish peroxidase (See FIG. 7 and FIG. 8, Panels D-O). Unlike thatfor G3BP-1, the immunohistochemistry showed no detectable expression ofG3BP-2 in normal lobes of the breast (See FIG. 8, Panel D) including thelobular and ductal epithelium and surrounding connective tissue. PanelsE and F of FIG. 8 show a higher magnification of two different ducts,Panel E shows a transverse section of a duct and Panel F shows alongitudinal section. As can be seen, there is no detectable expressionof G3BP-2 in normal ducts of the breast or within cells of thesurrounding connective tissue.

Immunohistochemistry revealed that G3BP-2 is over-expressed in breasttumours. FIG. 8, Panel G shows a normal duct adjacent to an IDC. As canbe seen by the brown staining, G3BP-2 is highly expressed in the tumourbut not expressed in the normal duct. This can be seen more clearly inPanel H which shows an IDC at higher magnification adjacent to a normalduct. Again, the normal duct does not express G3BP-2 and the IDC highlyexpresses G3BP-2.

The over-expression of G3BP-2 in breast tumours was not seen in allbreast tumours examined (12%). Panel I of FIG. 8 shows one case of IDCthat does not express G3BP-2. Another interesting observation noted whenexamining the expression of G3BP-2 in human breast tumours was that insome cases G3BP-2 is expressed in the nucleus of normal cells within theconnective tissues lying between the tumours (marked by the arrow), butnot in the cells within connective tissue away from the tumour (See FIG.8, Panel J).

FIG. 8, Panel K shows an example of a lower magnification of a tumourand adjacent connective tissue. As can be seen, G3BP-2 is expressed incells within the connective tissue peripheral to the tumour and theexpression becomes lower the further away the cells are from the tumour.These cells are most likely infiltrating lymphocytes as there seems tobe a greater population of these cells around the tumours. This couldsuggest that G3BP-2 expression is induced in response to a factorsecreted by some tumours or that G3BP-2 produces a chemotaxis-likeeffect.

Table 2 shows the results of all breast tumours examined for G3BP-2expression. Also listed is the available information on each of thebreast tumours including its oestrogen receptor status, tumour grade andstage. In summary, 88% of all tumours examined over-express G3BP-2 andno significant relationship was found between G3BP-2 over-expression andclinicopathological parameters of breast cancer such as stage, hormonereceptor status or nuclear or histological grade.

In the majority of human breast tumours that were screened, G3BP-2 isover-expressed and in many cases shows a distinct nuclear localisation(See FIG. 8, Panel M to O). Panels L to O show four different cases ofIDC with three different sub-cellular localisations. Panel L is anexample of a breast tumour where G3BP-2 is exclusively cytoplasmic.Panel M and N are two examples of breast tumours where G3BP-2 is foundin the nucleus and in the cytoplasm. Panel O shows G3BP-2 localisedaround the nuclear envelope region. These tumours also show acytoplasmic distribution for G3BP-2. This is the first case in whichG3BP-2 has been found in the nucleus in situ. Approximately 50% of alltumours that express G3BP-2 have G3BP-2 in the nucleus, although itshould be noted that it is possible that nuclear staining was notobserved in some cells where G3BP-2 is expressed at low levels, due tomasking by the haematoxylin counterstain. The nuclear staining variedbetween cases. Some cases had G3BP-2 in the nucleus of all cells,whereas others had less than 10% of cells with nuclear staining. Thepercentage of cells which express G3BP-2 in the nucleus does notcorrelate with the grade of the tumour or level of metastasis.

EXAMPLE 9

G3PB-2 Expression During the Cell Cycle

Interestingly, G3BP-2 can shuttle into the nucleus and its movementappears to be cell cycle dependent (See FIG. 8P-8T). In serum starvedresting cells G3BP-2 localises to the cytoplasm (FIG. 8P); however,within 2 hrs of releasing cells from G₀ G3BP-2 can be seen to move intothe nucleus (FIG. 8Q-8T) and at 5 hrs appears to be almost totallynuclear (FIG. 8R). After this time G3BP can be seen in both compartmentsconsistent with it shuttling between the nucleus and the cytoplasm(FIGS. 8S and 8T).

FIG. 8P-8T show the immunofluorescence of synchronised NIH 3T3 cells.Cells were synchronised by serum starvation and subsequently induced toenter the cell cycle by serum stimulation. Cells were stained for G3BP-2using immunofluorescent technique at several time intervals followingserum starvation. FIG. 8P shows the sub-cellular localisation of G3BP-2in cells in G₀ phase (time=0). The time after serum stimulation andhence cell cycle commencement is 2 hours, 5 hours, 9 hours and 12 hoursfor FIGS. 8Q, 8R, 8S and 8T, respectively.

Cell Cycle Synchronisation of NIH 3T3 Cells

Cell cycle synchronisation of NIH 3T3 cells was performed using theserum deprivation method (Tobey et al. 1988). NIH 3T3 cells were seededonto coverslips at sub-confluent conditions in 10% FCS. Following 24hours, the cells were washed 3 times with PBS and serum free medium wasthen replaced with medium containing 10% FCS. Coverslips were removedfrom the media during serum starvation, and at 2, 5, 9 and 12 hoursafter serum stimulation. The coverslips were then processed forimmunofluorescence to examine the expression of G3BP-2.

Immunofluorescence of Cultured Cells

NIH 3T3 cells were grown on coverslips, treated as described above, andwashed 3×2 min with PBS and dried overnight at room temperature. Thecells were fixed with 100% cold acetone for 5 min, allowed to dry, thenrehydrated by washing the coverslips with PBS 3×5 min. The cells werethen permeabilised by incubating the coverslips in 0.1% Triton-X 100 inPBS for 5 min. The detergent was then removed by washing the coverslips3×5 min with PBS. Primary antibody, diluted to the appropriateconcentration in PBS, was applied and left overnight at 4° C. Thefollowing morning the coverslips were washed 3×5 min in 1% normal goatserum (NGS)/1% bovine serum albumin (BSA) in PBS. Secondary antibody wasthen applied and incubated for 1 hour at room temperature. The secondaryantibody was anti-rabbit IgG conjugated with either a FITC or Rhodaminefluorescent tag (Molecular Probes, Eugene, USA) and was diluted in 0.1%Triton-X 100 in PBS at the dilution specified by the manufacturer. Thecoverslips were then washed 2×5 min in 0.1% Triton-X 100 in PBS followedby 2×5 min PBS. Finally, coverslips were mounted onto slides with 50%glycerol/50% PBS and sealed with nail polish. Images were generatedusing an Olympus Provis AX-70 and captured in digital format with aDAGE-MTI CCD camera using Scion Image 1.62 frame grabber software.Images were analysed using Adobe Photoshop 5 image-processing software(Adobe systems incorporated, Eastman Kodak Company, 1996).

EXAMPLE 10

Dendritic Cell Therapy

The invention provides pharmaceutical compositions and methods forpreventing or treating breast cancer in an animal. The pharmaceuticalcomposition comprises an isolated antigen presenting cell which has beenin contact with G3BP-2 polypeptide, fragment, homolog or derivativethereof, thereby loading or pulsing the cell with antigen. The isolatedantigen presenting cell is preferably a dendritic cell isolated from apatient which is undergoing dendritic cell therapy. The antigenpresenting cell may also be a precursor dendritic cell. The antigenpresenting cell may be cultured in vitro to expand or increase thenumber of cells before or after antigen loading. Alternatively, or inaddition, to loading the antigen presenting cell with G3BP-2polypeptide, fragment, homolog or derivative thereof, the antigenpresenting cell may be transfected with a nucleic acid encoding G3BP-2polypeptide, fragment, homolog or derivative thereof. The nucleic acidmay be DNA or RNA.

The method of preventing or treating breast cancer in an animal includesthe step of administering to the animal a pharmaceutical compositioncomprising antigen presenting cells which have been loaded or pulsedwith G3BP-2 polypeptide, fragment, homolog, or derivative thereof; orcells which have been transfected with a nucleic acid encoding G3BP-2,fragment, homolog, variant or derivative thereof.

In one embodiment, the method includes the steps of: (a) isolatingantigen presenting cells from an animal; (b) contacting the isolatedcells with G3PB-2 polypeptide, fragment, homolog, or derivative thereof,thereby antigen loading or pulsing the isolated cells; and (c)administering the loaded or pulsed isolated cells to the animal. Thecells are preferably autologous dendritic cells isolated from an animalwhich is administered the pharmaceutical composition.

In another embodiment, the invention method includes the steps of: (a)isolating antigen presenting cells from an animal; (b) transfecting theisolated cells with a nucleic acid encoding G3PB-2 polypeptide,fragment, homolog, or derivative thereof; and (c) administering thetransfected cells to the animal. The cells are preferably autologousdendritic cells isolated from an animal which is administered thepharmaceutical composition.

In either or both embodiments, the method may further include the stepof expanding the isolated antigen presenting cells in culture beforestep (c).

G3BP-2 and New Immunotherapy for Breast Cancer

Immuno-prevention is a very attractive therapy for breast cancer due tothe minimal tumor load and an ideal target for the intervention by theimmune system. One of the most promising strategies forimmuno-prophylaxic therapy is based on the use of dendritic cells (DC),the most potent antigen presenting cell (APC) of the immune system,responsible for the initiation of the immune response (Hart, 1997) andvital link between the innate and adaptive immunity (Clark et al.,2000). Researchers have investigated the important role of DC in healthand disease (Ho et al., 2001) and established mechanisms to optimizetheir APC capacity (Ho et al., 2002). Their potent capacity to inducecytotoxic T-lymphocyte (CTL) responses has been harnessed with successfor the treatment of various tumors (reviewed in (Lopez and Hart,2002)). Furthermore, it has been demonstrated that it is possible toobtain large number of DC from the blood (Lopez et al., 2002). This verypromising field of research still awaits critical inputs before beingapplied in a generic form.

In breast cancer research, the most important hurdle is the availabilityof tumor-associated antigens (TAA) that may elicit strong enough immuneresponses. An ideal TAA would have the characteristics of being crucialfor the development of cancer cells, over-(or selectively) expressed incancer cells, intracellularly localized and recognized by the immunesystem (Pardoll, 2002). Only two firm TAA candidates are available andthey are currently being tested. MUC-1, (CD227) is a transmembrane mucinmolecule normally polarised to the apical surface of epithelial cellscharacterized by a large extracellular domain of GC-rich random repeats(Gendler et al., 1991); it is highly expressed in breast cancer cellsand over-expressed in more than 90% of patients with breast cancer(Hadden, 1999). Her2/neu is a transmembrane glycoprotein, homologous tothe epidermal growth factor receptor that is over expressed in 20-30% ofpatients with breast cancer (Wang et al., 2001). It is correlated to theaggressiveness of the disease and an indicator of poor prognosis. Bothcellular and humoral immune responses to this protein have been detectedin patients (Wang and Hung, 2001). Additionally, TAA shared with othermalignancies, eg. MAGE 1 and 3 (melanoma antigens) are expressed in 20%and 26% of breast cancers respectively (Mashino et al., 2001; Otte etal., 2001; Russo et al., 1995). Finally, newer antigens such as thecarbohydrate antigen globo H (Gilewski et al., 2001) have emergedlately, and are currently being evaluated. None of the available TAAcandidates fulfill the abovementioned criteria for an optimal TAA inbreast cancer. The present researcher's recent data, however, indicatethat G3BP proteins represent excellent candidate TAA.

Immunogenicity of G3BP proteins within the context of the HLA-A*0201molecule have been evaluated, the most commonly found allele of theMajor Histocompatibility Complex (MHC), following thegenomic/immunogenic approach (Schultze and Vonderheide, 2001),incorporated herein by reference. Predicted HLA binding sequences wereidentified applying web-based algorithms (for example as provided bySYPEITHI (http://svfpeithi.bmi-heidelberg.com/) and BIMAS(http://bimas.dcrt.nih.gov/molbio/hla_bind/) and synthetic peptidesproduced for further evaluation. From the peptides generated,MHC/peptide binding assays demonstrated a very strong binding to theHLA-A*0201 molecule, as shown in FIG. 10. T2 cells were incubated withpeptide dilutions shown and HLA expression measured by flow cytometry.Two peptides (peptide 1 and 2) of G3BP-2 were tested and control matrixprotein influenza peptide 58-66 was used as a reference. Peptide 1 hasan amino acid sequence of KLPNFGFVV [SEQ ID NO:1] and peptide 2 has anamino acid sequence of IMFRGEVRL [SEQ ID NO:2]. Peptides 1 and 2 boundto the HLA-A*0201 molecule with an affinity equal to that of the controlinfluenza MP 58-66 peptide, a well-defined CTL epitope.

The various peptides were evaluated for the generation of CTL responsesinduced by DC in peripheral blood mononuclear cells (PBMC) from healthyindividuals and tested in ELISPOT (Gonzalez et al, 2000). T2 cells(target) pulsed with Peptide 1 or no peptide were labelled with ⁵¹Cr andincubated with clone IH7 (effector) at various ratios. After 4 hours,⁵¹Cr released from lysed cells was measured and the percentage ofspecific lysis calculated with the formula: 100×(experimentalrelease−spontaneous release)/(total release−spontaneous release). Totalrelease was obtained with detergent lysis of labelled target.

Strong responses were efficiently generated for Peptide 1, demonstratedby high frequencies of IFN-γ producing peptide specific CD8+ lymphocytesobtained after one (and two) round of stimulation in 2 separate donors.These results indicate that, indeed, the Peptide 1 epitope is includedin the T-cell receptor (TCR) repertoire and is, therefore, immunogenic.Confirming this finding, we generated Peptide 1 specific CTL clonescapable of identifying peptide-loaded targets in a classical chromiumrelease assay (FIG. 11), the desired profile for an efficient anti-tumorresponse (Lopez and Hart, 2002), incorporated herein by reference. Theseclones will be instrumental in the evaluation of the expression of thisantigen in cancer cells, allowing the formal evaluation of this moleculeas a TAA.

Throughout the specification the aim has been to describe the preferredembodiments of the invention without limiting the invention to any oneembodiment or specific collection of features. It will therefore beappreciated by those of skill in the art that, in light of the instantdisclosure, various modifications and changes can be made in theparticular embodiments exemplified without departing from the scope ofthe present invention.

The disclosure of each patent and scientific document, computer programand algorithm referred to in this specification is incorporated byreference in its entirety.

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1. An isolated G3BP-2 protein fragment comprising an NTF2-like domain,said isolated G3BP-2 protein fragment capable of binding another proteinby way of said NTF2-like domain.
 2. The isolated G3BP-2 protein fragmentof claim 1 wherein the isolated G3BP-2 protein fragment comprisesisolated G3BP-2a protein fragment and/or G3BP-2b protein fragment. 3.The isolated G3BP-2 protein fragment of claim 1 wherein said anotherprotein is selected from the group consisting of: ran nuclear porepolypeptide, ubiquitin hydrolase and GAP¹²⁰.
 4. The isolated G3BP-2protein fragment of claim 3 wherein the ubiquitin hydrolase is ODE1. 5.The isolated G3BP-2 protein fragment of claim 1 wherein the NTF2-likedomain comprises an amino acid sequence as set forth in SEQ ID NO: 22.6. An isolated protein complex comprising a G3BP-2 protein and a secondprotein bound to an NTF2-like domain of the G3BP-2 protein.
 7. Theisolated protein complex of claim 6 wherein said second protein isselected from the group consisting of: ran nuclear pore polypeptide,ubiquitin hydrolase and GAP¹²⁰.
 8. An isolated G3BP-2 protein, inclusiveof a fragment, homolog, variant or derivative used to elicit an immuneresponse in an animal.
 9. The isolated G3BP-2 protein of claim 8 whereinsaid animal is human.
 10. The isolated G3BP-2 protein of claim 8selected from the group (i) KLPNFGFW; [SEQ ID NO:1] (ii) IMFRGEVRL; [SEQID NO:2] and (iii) SATPPPAEPASLPQEPPKPRV [SEQ ID NO:3]


11. An isolated G3BP-2 protein fragment selected from the groupconsisting of: (a) KLPNFGFW; [SEQ ID NO:1] (b) IMFRGEVRL; [SEQ ID NO:2]and (c) SATPPPAEPASLPQEPPKPRV [SEQ ID NO:3]


12. An isolated nucleic acid encoding a protein of claim 1, inclusive offragments, homologs, variants and derivatives of said isolated protein.13. The isolated nucleic acid of claim 12 encoding a protein comprisingthe NTF2-like domain comprising an amino acid sequence as set forth inSEQ ID NO:
 22. 14. The isolated nucleic acid of claim 12 comprising asequence set forth in SEQ ID NO:
 23. 15. An isolated nucleic acidencoding the G3BP-2 protein fragment of claim
 10. 16. An expressionvector comprising the nucleic acid of claim 12 or claim
 14. 17. Use ofan antagonist to prevent or disrupt binding between G3BP-2 and anotherprotein.
 18. Use of the antagonist of claim 17, whereby said antagonistprevents or disrupts binding between a NTF2-like domain of G3BP-2 andsaid another protein.
 19. Use of the antagonist of claim 17 wherein saidantagonist is a mimetic of the NTF2-like domain of G3BP-2.
 20. Use ofthe antagonist of claim 17 wherein said antagonist binds to theNTF2-like domain.
 21. Use of the antagonist of claim 17 wherein saidantagonist is a protein.
 22. Use of the antagonist of claim 21 whereinsaid protein comprises an Src homology 3 (SH3) domain.
 23. Use of theantagonist of claim 22 wherein said protein comprises an amino acidsequence as set forth in SEQ ID NO:
 6. 24. Use of the antagonist ofclaim 17 wherein said antagonist is a non-peptide compound.
 25. Anisolated antigen presenting cell which has been contacted with a G3BP-2protein, fragment, homolog, variant or derivative thereof.
 26. Anisolated antigen presenting cell which has been transfected with anucleic acid encoding a G3BP-2 protein, inclusive of fragments,homologs, variants and derivatives thereof.
 27. The isolated antigenpresenting cell of claim 25 or claim 26 wherein said cell is a dendriticcell.
 28. The isolated antigen presenting cell of claim 25 or claim 26wherein said G3BP-2 protein, inclusive of a fragment, a homolog, avariant and a derivative thereof comprises an amino acid sequence as setforth in SEQ ID NO:
 5. 29. The isolated antigen presenting cell of claim25 or claim 26 wherein said G3BP-2 fragment comprises an amino acidsequence selected from the group consisting of: KLPNFGFVV [SEQ ID NO: 1]and IMFRGEVRL [SEQ ID NO: 2].
 30. An isolated lymphocyte that is G3BP-2antigen specific.
 31. The isolated lymphocyte of claim 30, wherein saidisolated lymphocyte is a cytotoxic T-lymphocyte.
 32. The isolatedlymphocyte cell of claim 30, wherein said G3BP-2 antigen is a protein,inclusive of fragments, homologs, variants and derivatives thereof,comprises an amino acid sequence as set forth in SEQ ID NO:
 5. 33. Theisolated lymphocyte of claim 30, wherein said G3BP-2 protein fragmentcomprises an amino acid sequence as set forth in SEQ ID NO: 1 or SEQ IDNO:
 2. 34. A pharmaceutical composition comprising at least one active,wherein the active is selected from the group consisting of: a proteinof any one of claims 1, 6, 11; a nucleic acid of any one of claims 12,15, 16 or an isolated antigen presenting cell or lymphocyte of any oneof claims 25, 26,
 30. 35. A method for preventing or treating breastcancer in a mammal including the step of administering to said mammal apharmaceutical composition comprising at least one active, wherein theactive is selected from the group consisting of: a protein of any one ofclaims 1, 6, 11; a nucleic acid of any one of claims 12, 15, 16, amimetic of the NTF2-like domain of G3BP-2; an antagonist that preventsor disrupts binding between a NTF2-like domain of G3BP-2 and anotherprotein; or isolated cell of any one of claims 25, 26,
 30. 36. Themethod of claim 35 wherein said mammal is human.
 37. A method formodulating cell proliferation including the step of administering to ananimal or isolated cell, an active which prevents or disrupts bindingbetween G3BP-2 and another protein.
 38. The method of claim 37 whereinsaid animal is human.
 39. A method for isolating a molecule that bindsG3BP-2, including the step of determining if one or more candidates in asample bind to the NTF2-like domain of G3BP-2.
 40. The method of claim39 wherein said molecule is an antagonist.
 41. The method of claim 40wherein said antagonist is a protein or a non-protein molecule.
 42. Amethod for diagnosing breast cancer in a mammal including the steps ofcomparing G3BP-2 protein expression in a test sample obtained from themammal with G3BP-2 in a reference sample, wherein if the expression ofG3BP-2 in the test sample is different than the reference sample, themammal is diagnosed with an increased likelihood of having breastcancer.
 43. The method of claim 42 when G3BP-2 protein expression isdetected using an antibody.
 44. The method of claim 43 wherein saidantibody binds to a G3BP-2 protein, inclusive of a fragment, a homolog,a variant and a derivative thereof, comprising an amino acid sequence asset forth in SEQ ID NO:
 5. 45. The method of claim 44 wherein saidG3BP-2 fragment comprises a NTF2-like domain.
 46. The method of claim 43wherein said antibody binds to a G3BP-2 protein fragment comprising anamino acid sequence SATPPPAEPASLPQEPPKPRV [SEQ ID NO: 3].
 47. The methodof claim 42 wherein said mammal is human.
 48. The method of claim 48wherein said test sample is breast tissue.
 49. A method for diagnosingbreast cancer in a mammal including the step of detecting a G3BP-2nucleic acid or fragment thereof in a test sample obtained from themammal.
 50. The method of claim 48 wherein said mammal is human.
 51. Amethod of immunising a mammal against breast cancer, including the stepof administering to said mammal an immunogenic agent comprising at leastone active selected from the group consisting of: (1) a G3BP-2 protein;(2) a fragment, a homolog, a variant or a derivative of (1); (3) aG3BP-2 nucleic acid; (4) a fragment, a homolog, a variant or aderivative of (3); (5) an isolated antigen presenting cell that has beencontacted with (1) or (2); and (6) an isolated antigen presenting cellthat has been transfected with a nucleic acid of (3) or (4).
 52. Themethod of claim 51 wherein said G3BP-2 protein fragment is selected fromthe group consisting of: KLPNFGFVV [SEQ ID NO: 1] and IMFRGEVRL [SEQ IDNO: 2].
 53. The method of claim 51 wherein said antigen presenting cellis a dendritic cell.
 54. The method of claim 51 wherein said mammal ishuman.